Tumor suppressor designated TS10q23.3

ABSTRACT

A specific region of chromosome 10 (10q23.3) has been implicated by series of studies to contain a tumor suppressor gene involved in gliomas, as well as a number of other human cancers. One gene within this region was identified, and the corresponding coding region of the gene represents a novel 47 kD protein. A domain of this product has an exact match to the conserved catalytic domain of protein tyrosine phosphatases, indicating a possible functional role in phosphorylation events. Sequence analyses demonstrated the a number of exons of the gene were deleted in tumor cell lines used to define the 10q23.3 region, leading to the classification of this gene as a tumor suppressor. Further analyses have demonstrated the presence of a number of mutations in the gene in both glioma and prostate carcinoma cells. Methods for diagnosing and treating cancers related to this tumor suppressor, designated as TS10q23.3, also are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is continuation application Ser. No. 10/299,003,filed 19 Nov. 2002, which is a divisional of application Ser. No.09/140,749 filed on 26 Aug. 1998 now U.S. Pat. No. 6,482,795, which inturn is a continuation-in-part of application Ser. No. 08/791,115, filed30 Jan. 1997 now U.S. Pat. No. 6,262,242. The present application isfurther related to and claims priority under 35 USC § 119(e) toprovisional patent application Ser. No. 60/057,750, filed 26 Aug. 1997and patent application Ser. No. 60/083,563, filed 30 Apr. 1998. All ofthese applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to the fields of oncology, genetics andmolecular biology. More particular the invention relates to theidentification, on human chromosome 10, of a tumor suppressor gene.Defects in this gene are associated with the development of cancers,such as gliomas.

II. Related Art

Oncogenesis was described by Foulds (1958) as a multistep biologicalprocess, which is presently known to occur by the accumulation ofgenetic damage. On a molecular level, the multistep process oftumorigenesis involves the disruption of both positive and negativeregulatory effectors (Weinberg, 1989). The molecular basis for humancolon carcinomas has been postulated, by Vogelstein and coworkers(1990), to involve a number of oncogenes, tumor suppressor genes andrepair genes. Similarly, defects leading to the development ofretinoblastoma have been linked to another tumor suppressor gene (Lee etal., 1987). Still other oncogenes and tumor suppressors have beenidentified in a variety of other malignancies. Unfortunately, thereremains an inadequate number of treatable cancers, and the effects ofcancer are catastrophic—over half a million deaths per year in theUnited States alone.

One example of the devastating nature of cancer involves tumors arisingfrom cells of the astrocytic lineage that are the most common primarytumors of the central nervous system (Russell & Rubinstein, 1989). Themajority of these tumors occur in the adult population. Primary braintumors also account for the most common solid cancer in the pediatricpatient population and the second leading cause of cancer deaths inchildren younger than 15 years of age. An estimated 18,500 new cases ofprimary brain tumors were diagnosed in 1994 (Boring et al., 1994).Epidemiological studies show that the incidence of brain tumors isincreasing and represents the third most common cause of cancer deathamong 18 to 35 year old patients. Due to their location within the brainand the typical infiltration of tumor cells into the surrounding tissue,successful therapeutic intervention for primary brain tumors often islimited. Unfortunately, about two-thirds of these afflicted individualswill succumb to the disease within two years. The most commonintracranial tumors in adults arise from cells of the glial lineage andoccur at an approximately frequency of 48% glioblastoma multiform (GBM),21% astrocytomas (A) (anaplastic (AA) and low grade) and 9% ependymomasand oligodendrogliomas (Levin et al., 1993).

Genetic studies have implicated several genes, and their correspondingprotein products, in the oncogenesis of primary brain tumors. Among thevarious reported alterations are: amplification of epidermal growthfactor receptor and one of its ligands, transforming growthfactor-alpha, N-myc; gli, altered splicing and expression of fibroblastgrowth factor receptors, and loss of function of p53, p16, Rb,neurofibromatosis genes 1 and 2, DCC, and putative tumor suppressorgenes on chromosomes 4, 10, 17 (non-p53), 19, 22, and X (Wong et al.,1987; El-Azouzi et al., 1989; Nishi et al., 1991; James et al., 1988;Kamb et al., 1984; Henson et al., 1994; Yamaguchi et al., 1994; Bianchiet al., 1994; Ransom et al., 1992; Rasheed et al., 1992; Scheck andCoons, 1993; Von Demling et al., 1994; Rubio et al., 1994; Ritland etal., 1995).

The most frequent alterations include amplification of EGF-receptor(˜40%), loss of function of p53 (˜50%), p16 (˜50%), Rb (˜30%) anddeletions on chromosome 10 (>90%). Furthermore, the grade or degree ofhistological malignancy of astrocytic tumors correlates with increasedaccumulation of genetic damage similar to colon carcinoma. Moreover,some changes appear to be relatively lineage- or grade-specific. Forinstance, losses to chromosome 19q occur predominantly inoligodendrogliomas, while deletions to chromosome 10 and amplificationand mutation of the EGF-receptor occur mainly in GBMs. The deletion ofan entire copy or segments of chromosome 10 is strongly indicated as themost common genetic event associated with the most common form ofprimary brain tumors, GBMs.

Cytogenetic and later allelic deletion studies on GBMs clearly havedemonstrated frequent and extensive molecular genetic alterationsassociated with chromosome 10 (Bigner et al., 1988; Ransom et al., 1992;Rasheed et al., 1992; James et al., 1988: Fujimoto et al., 1989; Fultset al., 1990, 1993; Karlbom et al., 1993; Rasheed et al., 1995; Sonodaet al., 1996; Albarosa et al., 1996). Cytogenetic analyses have clearlyshown the alteration of chromosome 10 as a common occurrence in GBMs,with 60% of tumors exhibiting alteration. Allelic deletion studies ofGBMs have also revealed very frequent allelic imbalances associated withchromosome 10 (90%). However, the losses are so extensive and frequentthat no chromosomal sublocalization of a consistent loss could beunequivocally defined by these analyses.

Several recent studies have implicated the region 10q25-26, specificallya 17 cM region from D10S190 to D10S216. A telomeric region from D10S587to D10S216 was implicated by allelic deletion analysis using a series oflow and high grade gliomas that exhibited only a partial loss ofchromosome 10 (Rasheed et al., 1995). This region (˜1 cM) was lost ornoninformative in 11 GBM's, 4 AA's, 1 A and 1 oligodendroglioma,suggesting localization of a candidate region. This study alsoillustrated that deletions to chromosome 10 occur in lower gradegliomas. Albarosa et al. (1996) suggest a centromeric candidate regionbased on a small allelic deletion in a pediatric brain tumor from themakers D10S221 to D10S209. Steck and Saya, using a series of GBMs, havesuggested two common regions of deletions, 10q26 and 10q24 (D10S192).

The short arm of chromosome 10 also has been implicated to containanother tumor suppressor gene. Studies first provided functionalevidence of a tumor suppressor gene on 10p in glioma (Steck et al.,1995) which was later shown for prostate (Sanchez et al., 1995; Murakamiet al., 1996). The latter study has implicated a 11 cM region betweenD10S1172 and D10S527. Allelic deletion studies of gliomas have shownextensive deletion on 10p, but again, no firm localization has beenachieved (Karlbom et al., 1993; Kimmelman et al., 1996; these regions ofchromosome 10 are shown to FIG. 1, below). Furthermore, theamplification of EGF-receptor has also been shown to occur almostexclusively in tumors that had deletions in chromosome 10, suggesting apossible link between these genetic alterations (Von Deimling et al.,1992).

Deletions on the long arm, particularly 10q24, also have been reportedfor prostate, renal, uterine, small-cell lung, endometrial carcinomas,meningioma and acute T-cell leukemias (Carter et al., 1990; Morita etal, 1991; Herbst et al., 1984; Jones et al., 1994; Rempel et al., 1993;Peiffer et al., 1995; Petersen et al., 1997). Recently, detailed studieson prostate carcinoma have shown that (1) the short and long arm ofchromosome 10 strongly appear to contain tumor suppressor genes, and (2)the localization of the long arm suppressor gene maps to the 10q23-24boundary (Gray et al., 1995; Ittmann, 1996, Trybus et al., 1996). Theregion of common deletion identified by these three groups is centeredaround D10S215 and extends about 10 cM (FIG. 1). The region overlapswith our candidate region, however, no further localization within theregion was reported fro prostate carcinoma. The allelic lossesassociated with prostate carcinoma also appear to occur in only about30-40% of the tumors examined. Furthermore, deletions are observed inmore advance staged tumors, similar to GBMs, and may be related tometastatic ability (Nihei et al., 1995; Komiya et al., 1996). Thecombination of these results suggest that multiple human cancersimplicate the region 10q23-24.

Differences in locations of the candidate regions suggest severalpossibilities. First, the presence of two or more tumor suppressor geneson 10q are possible. Second, not all deletions may effect the tumorsuppressor gene locus. These alternatives are not mutually exclusive. Insupport of the latter possibility, a potential latent centromere wassuggested to occur at 10q25 which may give rise to genetic alterations,particularly breakage (Vouillaire et al., 1993).

Despite all of this information, the identity of the gene (or genes)involved with the 10q23-24-related tumor suppression remained elusive.Without identification of a specific gene and deduction of the proteinfor which it codes, it is impossible to begin developing an effectivetherapy targeting this product. Thus, it is an important goal to isolatethe tumor suppressor(s) located in this region and determine itsstructure and function.

SUMMARY OF THE INVENTION

Therefore, it is an objective of the present invention to provide atumor suppressor, designated as TS10q23.3 (also referred to as MMAC orPTEN). It also is an objective to provide DNAs representing all or partof a gene encoding TS10q23.3. It also is an objective to providesmethods for using these compositions.

In accordance with the foregoing objectives, there is provided, in oneembodiment, a tumor suppressor designated as TS10q23.3. The polypeptidehas, in one example, the amino acid sequence as set forth in SEQ IDNO:2; SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:49, SEQ ID NO:55 or SEQ IDNO:57. In a further example, the polypeptide has the amino acid sequenceas set forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or SEQ IDNO:63, Also provided is an isolated peptide having between about 10 andabout 50 consecutive residues of a tumor suppressor designated asTS10q23.3. The peptide may be conjugated to a carrier molecule, forexample, KLH or BSA.

In another embodiment, there is provided a monoclonal antibody thatbinds immunologically to a tumor suppressor designated as TS10q23.3. Theantibody may be non-cross reactive with other human polypeptides, or itmay bind to non-human TS10q23.3, but not to human TS10q23.3. Theantibody may further comprise a detectable label, such as a fluorescentlabel, a chemiluminescent label, a radiolabel or an enzyme. Alsoencompassed are hybridoma cells and cell lines producing suchantibodies.

In another embodiment, there is included a polyclonal antisera,antibodies of which bind immunologically to a tumor suppressordesignated as TS10q23.3. The antisera may be derived from any animal,but preferably is from other than human, mouse or dog.

In still another embodiment, there is provided an isolated nucleic acidcomprising a region, or the complement thereof, encoding a tumorsuppressor designated TS10q23.3 or an allelic variant or mutant thereof.The tumor suppressor coding region may be derived from any mammal but,in particular embodiments, is selected from murine, canine and humansequences. Mutations include deletion mutants, insertion mutants,frameshift mutants, nonsense mutants, missense mutants or splicemutants. In certain embodiments, the mutation comprises a homozygousdeletion of one or more of the exons of the tumor suppressor. Inspecific embodiments, exons 3, 4, 5, 6, 7, 8, or 9 are deleted. In otherembodiments exon 2 is deleted. In certain embodiments all of exons 3-9are deleted. In other embodiments, exons 2-9 are deleted. In aparticular embodiment, the tumor suppressor has the amino acid sequenceof SEQ ID NO:2; SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:49, SEQ ID NO:55or SEQ ID NO:57. The nucleic acid may have the sequence set forth in SEQID NO:1, SEQ ID NO:9, SEQ ID NO:16, SEQ ID NO:54, or SEQ ID NO:56 or acomplement thereof. The nucleic acid may further have the sequence setforth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27 or acomplement thereof. The nucleic acid may also have the sequence setforth in SEQ ID NO:64 or a complement thereof. The nucleic acid may begenomic DNA, complementary DNA or RNA.

In certain embodiments, the mutant is a splice mutant. In particularembodiments, the splice mutation is in exon 3, exon 8 or intron 2. Inmore specific embodiments, the splice mutation results in (i) a changefrom G to T at position +1 in exon 3, or (ii) a change from G to T atposition +1 in exon 8 or (iii) a change from G to T at position −1 inintron 2.

In certain other embodiments, the mutant is a missense mutant. Inparticular embodiments, the missense mutation is in exon 2. In morespecific embodiments, the missense mutation results in a change from Tto G at position 46 of exon 2, leading to a change from LEU to ARG. Incertain other embodiments, the missense mutation results in a changefrom G to A at position 28 of exon 2, leading to a change from a GLY toa GLU. In certain other embodiments, the mutation results in a changefrom C to T at position 53 of exon 2. In certain other embodiments, themissense mutation results in a change from CC to TT at positions 112 and113 of exon 2, leading to a change from PRO to PHE at amino acid 38 ofsaid tumor suppressor. In certain embodiments, the missense mutation isin exon 5. In specific embodiments, the missense mutation may results ina change from T to G at position 323 of exon 5, leading to a change fromLEU to ARG at amino acid 108 of said tumor suppressor. In other specificembodiments, the missense mutation results in a change from T to C atposition 331 of exon 5 leading to a change from TRP to ARG at amino acid111 of said tumor suppressor. In certain other embodiments, the missensemutation results in a change from T to G at position 335 of exon 5leading to a change from LEU to ARG at amino acid 112 of said tumorsuppressor. In still other embodiments, the missense mutation results ina change from G to A at position 407 of exon 5, leading to a change fromCYS to TYR at amino acid 136 of said tumor suppressor. In otherexemplary missense embodiments, the missense mutation results in achange from T to C at position 455 of exon 5, leading to a change fromLEU to PRO at amino acid 152 of said tumor suppressor. In yet otherembodiments, the missense mutation is in exon 6. More specifically, themissense mutation results in a change from C to T at position 517 ofexon 6, leading to a change from ARG to CYS at amino acid 173 of saidtumor suppressor. In other specific embodiments, the missense mutationresults in a change from G to C at position 518 of exon 6 leading to achange from ARG to a PRO at amino acid 173 of said tumor suppressor.

Yet other embodiments provide a nonsense mutant. In certain embodiments,the nonsense mutation is in exon 5. More specifically, the nonsensemutation results in a change from C to T at position 388 of exon 5,leading to a change from ARG to a STOP at codon 130 of said tumorsuppressor. In other embodiments, the nonsense mutation is in exon 7.More specifically, the nonsense mutation results in a change from C to Tat position 697 of exon 7, leading to a change from ARG to a STOP atcodon 233 of said tumor suppressor. In certain embodiments, the nonsensemutation is in exon 8. More specifically, the nonsense mutation resultsin a change from C to T at position 202 of exon 8.

In still further embodiments of the present invention, there iscontemplated a frameshift mutant. In particular embodiments, theframshift mutation is in exon 7. More specifically, the frameshiftmutation is a deletion of A at position 705 of exon 7, leading to atruncated tumor suppressor expression. In particular embodiments,frameshift mutation results is a deletion of G at position 823 of exon7, leading to a truncated tumor suppressor expression. In otherembodiments, the frameshift mutation is an insertion of TT at position98 in exon 7. In certain embodiments, the frameshift mutation is inexon 1. More specifically, the frameshift mutation is a deletion of AAat positions 16 and 17 of exon 1.

In additional embodiments, the nucleic acid comprises a complementaryDNA and further comprises a promoter operably linked to the region, orthe complement thereof, encoding the tumor suppressor. Additionalelements include a polyadenylation signal and an origin of replication.

Viral vectors such as retrovirus, adenovirus, herpesvirus, vacciniavirus and adeno-associated virus also may be employed. The vector may be“naked” or packaged in a virus particle. Alternatively, the nucleic acidmay comprise an expression vector packaged in a liposome.

Various sizes of nucleic acids are contemplated, but are not limiting:about 1212 bases, about 1500 bases, about 2000 bases, about 3500 bases,about 5000 bases, about 10,000 bases, about 15,000 bases, about 20,000bases, about 25,000 bases, about 30,000 bases, about 35,000 bases, about40,000 bases, about 45,000 bases, about 50,000 bases, about 75,000 basesand about 100,000 bases.

In yet another embodiment, there is provided an isolated oligonucleotideof between about 10 and about 50 consecutive bases of a nucleic acid, orcomplementary thereto, encoding a tumor suppressor designated asTS10q23.3. The oligonucleotide may be about 15 bases in length, about 17bases in length, about 20 bases in length, about 25 bases in length orabout 50 bases in length.

In another embodiment, there is provided a method of diagnosing a cancercomprising the steps of (i) obtaining a sample from a subject; and (ii)determining the expression a functional TS10q23.3 tumor suppressor incells of the sample. The cancer may be brain, lung, liver, spleen,kidney, lymph node, small intestine, pancreas, blood cells, colon,stomach, breast, endometrium, prostate, testicle, ovary, skin, head andneck, esophagus, bone marrow and blood cancer. In preferred embodiments,the cancer is prostate cancer or breast cancer. In another preferredembodiment, cancer is a brain cancer, for example, a glioma. The sampleis a tissue or fluid sample.

In one format, the method involves assaying for a nucleic acid from thesample. The method may further comprise subjecting the sample toconditions suitable to amplify the nucleic acid. Alternatively, themethod may comprise contacting the sample with an antibody that bindsimmunologically to a TS10q23.3, for example, in an ELISA. Thecomparison, regardless of format, may include comparing the expressionof TS10q23.3 with the expression of TS10q23.3 in non-cancer samples. Thecomparison may look at levels of TS10q23.3 expression. Alternatively,the comparison may involve evaluating the structure of the TS10q23.3gene, protein or transcript. Such formats may include sequencing,wild-type oligonucleotide hybridization, mutant oligonucleotidehybridization, SSCP™ and RNase protection. Particular embodimentsinclude evaluating wild-type or mutant oligonucleotide hybridizationwhere the oligonucleotide is configured in an array on a chip or wafer.

In another embodiment, there is provided a method for altering thephenotype of a tumor cell comprising the step of contacting the cellwith a tumor suppressor designated TS10q23.3 under conditions permittingthe uptake of the tumor suppressor by the tumor cell. The tumor cell maybe derived from a tissue such as brain, lung, liver, spleen, kidney,lymph node, small intestine, blood cells, pancreas, colon, stomach,breast, endometrium, prostate, testicle, ovary, skin, head and neck,esophagus, bone marrow and blood tissue. The phenotype may be selectedfrom proliferation, migration, contact inhibition, soft agar growth orcell cycling. The tumor suppressor may be encapsulated in a liposome orfree.

In another embodiment, there is provided a method for altering thephenotype of a tumor cell comprising the step of contacting the cellwith a nucleic acid (i) encoding a tumor suppressor designated TS10q23.3and (ii) a promoter active in the tumor cell, wherein the promoter isoperably linked to the region encoding the tumor suppressor, underconditions permitting the uptake of the nucleic acid by the tumor cell.The phenotype may be proliferation, migration, contact inhibition, softagar growth or cell cycling. The nucleic acid may be encapsulated in aliposome. If the nucleic acid is a viral vector such as retrovirus,adenovirus, adeno-associated virus, vaccinia virus and herpesvirus, itmay be encapsulated in a viral particle.

In a further embodiment, there is provided a method for treating cancercomprising the step of contacting a tumor cell within a subject with atumor suppressor designated TS10q23.3 under conditions permitting theuptake of the tumor suppressor by the tumor cell. The method may involvetreating a human subject.

In still a further embodiment, there is provided a method for treatingcancer comprising the step of contacting a tumor cell within a subjectwith a nucleic acid (i) encoding a tumor suppressor designated TS10q23.3and (ii) a promoter active in the tumor cell, wherein the promoter isoperably linked to the region encoding the tumor suppressor, underconditions permitting the uptake of the nucleic acid by the tumor cell.The subject may be a human.

In still yet a further embodiment, there is provided transgenic mammalin which both copies of the gene encoding TS10q23.3 are interrupted orreplaced with another gene.

In an additional embodiment, there is provided a method of determiningthe stage of cancer comprising the steps of (i) obtaining a sample froma subject; and (ii) determining the expression a functional TS10q23.3tumor suppressor in cells of the sample. The cancer may be a braincancer and the stage is distinguished between low grade and glioma. Thedetermining may comprise assaying for a TS10q23.3 nucleic acid orpolypeptide in the sample.

In yet an additional example, there is provided a method of predictingtumor metastasis comprising the steps of (i) obtaining a sample from asubject; and (ii) determining the expression a functional TS10q23.3tumor suppressor in cells of the sample. The cancer may be distinguishedas metastatic and non-metastatic. The determining may comprise assayingfor a TS10q23.3 nucleic acid or polypeptide in the sample.

In still yet an additional embodiment, there is provided a method ofscreening a candidate substance for anti-tumor activity comprising thesteps of (i) providing a cell lacking functional TS10q23.3 polypeptide;(ii) contacting the cell with the candidate substance; and (iii)determining the effect of the candidate substance on the cell. The cellmay be a tumor cell, for example, a tumor cell having a mutation in thecoding region of TS10q23.3.7. The mutation may be a deletion mutant, aninsertion mutant, a frameshift mutant, a nonsense mutant, a missensemutant or splice mutant. The determining may comprise comparing one ormore characteristics of the cell in the presence of the candidatesubstance with characteristics of a cell in the absence of the candidatesubstance. The characteristic may be TS10q23.3 expression, phosphataseactivity, proliferation, metastasis, contact inhibition, soft agargrowth, cell cycle regulation, tumor formation, tumor progression andtissue invasion. The candidate substance may be a chemotherapeutic orradiotherapeutic agent or be selected from a small molecule library. Thecell may be contacted in vitro or in vivo.

In still a further additional embodiment, there is provided a method ofscreening a candidate substance for anti-kinase activity comprising thesteps of (i) providing a having TS10q23.3 polypeptide comprising atleast one tyrosine kinase site; (ii) contacting the cell with thecandidate substance; and (iii) determining the effect of the candidatesubstance on the phosphorylation of the site. The determining maycomprise comparing one or more characteristics of the cell in thepresence of the candidate substance with characteristics of a cell inthe absence of the candidate substance. The characteristic may bephosphorylation status of TS10q23.3, TS10q23.3 expression, phosphataseactivity, proliferation, metastasis, contact inhibition, soft agargrowth, cell cycle regulation, tumor formation, tumor progression andtissue invasion. The candidate substance may be a chemotherapeutic orradiotherapeutic agent or be selected from a small molecule library. Thecell may be contacted in vitro or in vivo.

In yet another embodiment, the present invention provides a method ofdiagnosing Cowden's Syndrome comprising the steps of obtaining a samplefrom a subject; and determining the expression a functional TS10q23.3gene product in cells of the sample. In particularly preferredembodiments, the cells may be selected from the group consisting ofbreast, ovarian, thyroid and endometrial cells. In other embodiments,the sample may be a tissue or fluid sample. In other aspects of theinvention the determining comprises assaying for a nucleic acid from thesample. In more preferred aspects, the method may further comprisesubjecting the sample to conditions suitable to amplify the nucleicacid. In other embodiments, the method may further comprise the step ofcomparing the expression of TS10q23.3 with the expression of TS10q23.3in non-Cowden's Syndrome samples. In particular embodiments, thecomparison may involve evaluating the level of TS10q23.3 expression. Inmore particular embodiments, the Cowden's Syndrome sample comprises amutation in the coding sequence of TS10q23.3. The mutation may be aframeshift mutation, a deletion mutation, an insertion mutation or amissense mutation. In more particular embodiments the mutation is inexon 7. In other particular embodiments, the mutation results in apremature termination of the TS10q23.3 gene product. In otherembodiments, the deletion mutation is in exon 8. In certain embodimentsthe insertion is in exon 2. In particularly preferred embodiments, themutation is an insertion of AT at base 791 of exon 7. In otherparticularly preferred embodiments, the mutation is a thirteen base pairdeletion at base 915 of exon 8. In another preferred embodiment, themutation is a three base pair insertion at base 137 of exon 2. Morespecifically the three base pair insertion results encodes for an ASN inthe TS10q23.3 gene product.

In a further aspect, there is also provided a method of diagnosing asubject predisposed to breast cancer comprising the steps of obtaining asample from a subject; and determining the expression a functionalTS10q23.3 gene product in cells of the sample. In particularembodiments, the cells may be selected from the group consisting ofbreast, ovarian cells, thyroid cells and endometrial cells. In otherembodiments, the sample is a tissue or fluid sample. In a particularlypreferred aspect the method further comprises the step of comparing theexpression of TS10q23.3 with the expression of TS10q23.3 in normalsamples. In more defined aspects the sample comprises a mutation in thecoding sequence of TS10q23.3. The mutation may be a frameshift mutation,a deletion mutation, an insertion mutation or a missense mutation. Inmore particular embodiments the mutation is in exon 7. In otherparticular embodiments, the mutation results in a premature terminationof the TS10q23.3 gene product. In other embodiments, the deletionmutation is in exon 8. In certain embodiments the insertion is in exon2. In particularly preferred embodiments, the mutation is an insertionof AT at base 791 of exon 7. In other particularly preferredembodiments, the mutation is a thirteen base pair deletion at base 915of exon 8. In another preferred embodiment, the mutation is a three basepair insertion at base 137 of exon 2. More specifically the three basepair insertion results encodes for an ASN in the TS10q23.3 gene product.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1.—Localization of Candidate Tumor Suppressor Loci on HumanChromosome 10. Various loci on the human chromosome 10 have beenimplicated as possible sites for tumor suppressing activity. Theselocations, and the reporting group, are depicted.

FIG. 2.—Illustration of Homozygous Deletions in Glioma Cell Lines.Various glioma cell lines were screened for the presence of deletions inboth copies of loci on chromosome 10. Loci are indicated on the verticalaxis and cell lines are listed across the horizontal axis. Homozygousloss is indicated by a darkened oval. The glioma cell lines D54, EFC-2,A172 and LG11 were examined for the presence of marker AFMA086WG9(AFM086). The marker was shown to be deleted in multiplexed polymerasechain reactions using several additional chromosome 10 polymorphicalleles in independent reactions. Allele D10S196 is shown as the controlfor the PCR™ reaction. EFC-2 cells showed homozygous deletion of 4contiguous markers (see FIG. 2).

FIG. 3.—Illustration of Regions of Chromosome 10: Presence or Absence ofDNA Microsatellite Markers in Hybrid Clone. Regions of chromosome 10presence (solid circle) or absence (open circle) of DNA corresponding tochromosome 10 specific microsatellite markers from eleven subclones ofthe somatic cell hybrid clone U251.N10.7 that were transferred intomouse A9 cells are illustrated. The U251.N10.6 and U251.N10.8 somaticcell hybrids are fully suppressed clones, exhibiting no or little growthin soft agarose, and the U251.10.5A and C subclones are partiallysuppressed (Steck et al., 1995). The difference between the fullysuppressed clones and the partially suppressed clones provides afunctional localization of the tumor suppressor gene. The possibleregions that contain the tumor suppressor gene are indicated by thehatched boxes. The hatched box at 10q23.3 overlaps with the homozygousdeletions and region implicated by allelic deletion analysis (see FIG. 2and FIG. 4).

FIG. 4.—Deletion Map of Chromosome 10 in Human Gliomas. The regionbounded by the markers D10S551 to D10S583 are located in a 10 cM region.The microsatellites are shown in their order of most probably linkageand mapped to their approximate chromosomal location based on theradiation hybrid map as described by Gyapay et al., 1994. The region ofchromosome 101 that is not involved in anaplastic astrocytomas and oneglioma is shown in the boxed regions of the tumor. The critical regiondefined from the homozyogous deletion analysis and not excluded by thisanalysis is shown by the solid bar on the right side.

FIG. 5.—Mapping of BAC106d16. Mapping of the BAC designated 106d16, anddemonstration of homozygous deletion by Southern blotting isillustrated. The partial restriction map of 106d16 is depicted. Theillustration of the blot shows the homozygous deletion of Eco band #14(Mr approx. 11 kb) in EFC-2 cells.

FIG. 6.—Coding Sequence and 5′- and 3′-Flanking Regions of TS10q23.3.Coding region is in bold as is the first in frame stop codon.

FIG. 7.—Predicted Amino Acid Sequence of TS10q23.3 Product.Abbreviations are A, alanine; C, cysteine; D, aspartic acid; E, glutamicacid; F; phenylalanine; G, glycine; H, histidine; I, isoleucine; K,lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q,glutamine; R, arginine; S, serine; T, threonine; V, valine; W,tryptophan; Y, tyrosine. Phosphatase consensus site is in bold; tyrosinephosphorylation sites are italicized and underlined.

FIG. 8.—Deletional Analysis of 10q23.3. Glioma line initially indicatedas having homozygous deletions in 10q23.3 were reanalyzed for thepresence of the TS10q23.3 gene. Darkened oval indicates that the generegion is present; open oval indicates a homozygous deletion in the generegion. *—indicates exons trapped.

FIG. 9.—Homology Comparison of Human TS10q23.3 with Mouse and DogHomologs. The initiation ATG codon and methionine amino acid aredesignated at the start (1) position. The termination codon is TGA(1210). Alterations between the human and mouse or dog sequences on thegenomic or amino acid level are designated by a star in the sequencecompared. The dog and human amino acid sequences are identical; themouse sequence differed at position 398, where the mouse has a Serine,as opposed to a Threonine in dog and human.

FIG. 10.—Sequence of Exons and Surrounding Intronic Regions ofTS10q23.3. The exons are denoted as capital letters starting at positionone, and introns are designated lower case letters; except for the firstexon where the initiation codon starts at position one and the 3′exon/intron boundary is at position 79 and 80, respectively. The lowercase letter designate (Table 5) corresponds to the numbering of thesequence presented in this figure, except for the first exon. Themutations for U87 and U138 are at the first intron G residue [G+1>T]after the exon (exon 7 and 8, respectively). For T98G and KE, the pointmutations are at positions 46 and 28 of exon 2, respectively. For LnCapcells, the mutation is a deletion of bases 16 and 17 in the firstintron.

FIGS. 11A-G.—Analysis of Secondary Structures in TS1023.3. FIG. 11A:Hydrophilicity plot; FIG. 11B: Surface probability plot; FIG. 11C:Flexibility plot; FIG. 11D: Antigenic index plot; FIG. 11E: Amphiphilichelix plot; FIG. 11F: Amphiphilic sheet plot; FIG. 11G: Secondarystructure plot.

FIGS. 12A-I.—Comparison of Predicted Characteristics in TS10q23.3 andPoint Mutants T98G and KE. FIG. 12A: Hydrophilicity plot of residues1-60 of wild-type polypeptide; FIG. 12B: Surface probability plot ofresidues 1-60 of wild-type polypeptide; FIG. 12C: Secondary structureplot of residues 1-60 of wild-type polypeptide; FIG. 12D: Hydrophilicityplot of residues 1-60 of KE mutant; FIG. 12E: Surface probability plotof residues 1-60 of KE mutant; FIG. 12F: Secondary structure plot ofresidues 1-60 of KE mutant; FIG. 12G: Hydrophilicity plot of residues1-60 of T98G mutant; FIG. 12H: Surface probability plot of residues 1-60of T98G mutant; FIG. 12I: Secondary structure plot of residues 1-60 ofT98G mutant. The T98G mutation (Leu→Arg) at residue 42 results in theloss of proposed helix secondary structure of TS10q23.3. The mutation inKE at residue 36 (Gly→Glu) would significantly increase the length ofthe proposed helical structure in the region. Both mutations wouldaffect the same helical structure. Also, minor changes in thehydrophilicity and surface probability arise.

FIG. 13A. Homozygous deletion of the TS10Q23.3 gene in human tumor celllines and TS10Q23.3 mRNA expression levels in primary glioblastomas.Shown are four cell lines, breast carcinoma TCL11A11, melanoma TCL11D7,melanoma TCL11D9 and leukemia TCL10G9 (control sample withouthomozygously deleted TS10Q23.3), each examined by PCR™ amplificationusing the following sequence tagged sites: (1) TS10Q23.3 exon 1, (2)TS10Q23.3 exon 2, (3) TS10Q23.3 exon 3, (4) TS10Q23.3 exon 4, (5)TS10Q23.3 exon 5, (6) TS10Q23.3 exon 6, (7) TS10Q23.3 exon 7, (8)TS10Q23.3 exon 8, (9) TS10Q23.3 exon 9, (10) control MKK4 exon 8.

FIG. 13B. Homozygous deletion of the TS10Q23.3 gene in human tumor celllines and TS10Q23.3 mRNA expression levels in primary glioblastomas.Schematic of the homozygous deletions observed in the TS10Q23.3 gene ofTCLs screened. Closed circles represent exons that are not homozygouslydeleted while open circles represent exons that are lost.

FIG. 13C. Homozygous deletion of the TS10Q23.3 gene in human tumor celllines and TS10Q23.3 mRNA expression levels in primary glioblastomas.Expression of TS10Q23.3 message in human normal brain and GBM specimensas detected by RT-PCR™ analysis. The 5′ terminal amplicon of TS10Q23.3is shown. The lanes shown include a control amplicon (C) from PL-1 lowgrade glioma cDNA, along with seven normal and tumor specimens. Six ofthe 10 GBMs examined were examined for LOH surrounding the TS10Q23.3locus and TS10Q23.3 gene alterations. All six samples exhibited LOH butno mutations were detected when the inventors screened their DNAs bysequencing. The expression levels of GADPH message was used to controlfor equivalent template quantities and qualities.

FIG. 13D. Homozygous deletion of the TS10Q23.3 gene in human tumor celllines and TS10Q23.3 mRNA expression levels in primary glioblastomas.Ratio of the RT-PCR™ amplicon intensities of TS10Q23.3 to GADPH forevery normal and GBM specimen.

FIG. 14. Representation of the putative functional domains of TS10Q23.3and the location of identified alterations. The N-terminal half ofTS10Q23.3 is homologous to phosphatases, as well as the cytoskeletalproteins, tensin and auxilin (brown box). Also shown are the locationsof the core phosphatase domain (red box), three potential tyrosinephosphorylation sites (blue boxes) and two potential serinephosphorylation sites (yellow boxes). The PDZ motif, ITKV, is located atthe C-terminus of the protein. Shown are TS10Q23.3 variants identifiedby Steck et al. (1997), Li et al., (1997), and Liaw et al. (1997), andalterations detected in this study; blue arrows mark missensesubstitutions, black arrows indicate in-frame insertions or deletions,green arrows mark potential splicing variants, and red arrows representframeshift or nonsense mutations that result in TS10Q23.3 truncations.Asterisks indicate germline mutations that were detected in Cowden'spatients (Liaw et al., 1997), while the closed circles indicate lesionsthat have been observed in two presumably independent DNA samples.

FIG. 15. Haplotype construction with markers on chromosome 10 in fourfamilies with CS.

FIG. 16. DNA Sequencing of TS10Q23.3 in a family with CS and early onsetbreast cancer. The affected mother (black circle) demonstrates a 2 basepair insertion (AT) in exon 5, which is not seen in her unaffectedbrother (open square). Her affected daughter has inherited the ATinsertion.

FIG. 17. Exogenous MMAC1 protein expression. U87MG cells were infectedwith MMCB or GFCB at the indicated concentrations (particle numbers/ml)for 24 hr, then lysates were prepared immediately (24 hr) or 24 hr later(48 hr). Western blotting was performed as described in Methods. Proteinsize markers are shown at left. MMAC1 protein migrated at approximately55 kD in agreement with Li et al., 1997.

FIG. 18. FACS infectivity assay. U87MG cells were infected with GFCB atthe indicator/concentrations for 24 hr. The fraction of cells expressinggreen fluorescent protein was quantitated by flow cytometry. pn/ml:adenovirus particle numbers per ml.

FIG. 19A and FIG. 19B. Inhibition of in vitro proliferation by MMCB.FIG. 19A. ³H-thymidine uptake. FIG. 19B. Viable cell count assay. Errorbars are S.D. (3 replicates). Pn/ml: adenovirus particle numbers per ml.

FIG. 20. Soft-agar colony formation. U87MG cells were infected withGFCB, MMCB or FTCB at the indicated concentrations for 24 hr. Meancolony numbers±S.D. are plotted. pn/ml: adenovirus particle numbers perml.

SEQUENCE SUMMARY

SEQ ID NO:1=human TS10q23.3 gene sequence (FIGS. 6 and 9); SEQ IDNO:2=human TS10q23.3 peptide sequence from CDS of SEQ ID NO:1; SEQ IDNO:3=translation of bases 3-119 of SEQ ID NO:1; SEQ ID NO:4=translationof bases 123-242 of SEQ ID NO:1; SEQ ID NO:5=translation of bases246-272 of SEQ ID NO:1; SEQ ID NO:6=translation of bases 276-317 of SEQID NO:1; SEQ ID NO:7=translation of bases 321-449 of SEQ ID NO:1; SEQ IDNO:8=translation of bases 453-2243 of SEQ ID NO:1; SEQ ID NO:9=mouseTS10q23.3 gene sequence (FIG. 9); SEQ ID NO:10=mouse TS10q23.3 peptidesequence from CDS of SEQ ID NO:9; SEQ ID NO:11=translation of bases14-55 of SEQ ID NO:9; SEQ ID NO:12=translation of bases 59-166 of SEQ IDNO:9; SEQ ID NO:13=translation of bases 172-222 of SEQ ID NO:9; SEQ IDNO:14=translation of bases 223-273 of SEQ ID NO:9; SEQ IDNO:15=translation of bases 283-1959 of SEQ ID NO:9; SEQ ID NO:16=dogTS10q23.3 gene sequence (FIG. 9); SEQ ID NO:17=dog TS10q23.3 peptidesequence from CDS of SEQ ID NO:16; SEQ ID NO:18=translation of bases1-1290 of SEQ ID NO:16; SEQ ID NO:19=exon 1 (FIG. 10); SEQ ID NO:20=exon2 (FIG. 10); SEQ ID NO:21=exon 3 (FIG. 10); SEQ ID NO:22=exon 4 (FIG.10); SEQ ID NO:23=exon 5 (FIG. 10); SEQ ID NO:24=exon 6 (FIG. 10); SEQID NO:25=exon 7 (FIG. 10); SEQ ID NO:26=exon 8 (FIG. 10); SEQ IDNO:27=exon 9 (FIG. 10); SEQ ID NO:28=a motif from residues 88 to 98; SEQID NO:29=conserved catalytic domain of a protein tyrosine phosphatase(Denu et al., 1996); SEQ ID NO:30=residues 1-60 of wild-type TS10q23.3polypeptide (FIGS. 12A-12C); SEQ ID NO:31=residues 1-60 of T98G mutantTS10q23.3 polypeptide (FIGS. 12D-12F); SEQ ID NO:32=residues 1-60 of KEmutant TS10q23.3 polypeptide (FIGS. 12G-12I); SEQ ID NO:33=CA6.ex8.FBprimer; SEQ ID NO:34=CA6.ex8.RQ primer; SEQ ID NO:35: =CA6.ex8.FCprimer; SEQ ID NO:36=CA6.ex8.RR primer; SEQ ID NO:37=nested primer usedto obtain secondary amplicons exon 8 FB-RQ; SEQ ID NO:38=nested primerused to obtain secondary amplicons exon 9 FB-RR; SEQ ID NO:39=M5′Fprimer; SEQ ID NO:40=M5′ R primer; SEQ ID NO:41=M3′F primer; SEQ IDNO:42:=F3′R primer; SEQ ID NO:43=primer in first round PCR™ in humanfetal brain; SEQ ID NO:44=primer in first round PCR™ in human fetalbrain; SEQ ID NO:45=primer in second round PCR™ in human fetal brain;SEQ ID NO:46=primer in second round PCR™ in human fetal brain; SEQ IDNO:47=primer used to generate a specific 303 bp product from thepseudogene and not TS10q23; SEQ ID NO:48=primer used to generate aspecific 303 bp product from the pseudogene and not TS10q23; SEQ IDNO:49=mouse MMAC1 protein sequence; SEQ ID NO:50=peptide sequence; SEQID NO:51=translation of bases 321-1034 of SEQ ID NO:1; SEQ IDNO:52=translation of bases 169-750 of SEQ ID NO:9; SEQ IDNO:53=translation of bases 1-108 of SEQ ID NO:16; SEQ ID NO:54=dog MMAC1gene sequence; SEQ ID NO:55=dog MMAC1 protein sequence from CDS of SEQID NO:54; SEQ ID NO:56=mouse MMAC gene sequence; SEQ ID NO:57=mouseMMAC1 protein sequence from CDS of SEQ ID NO:56; SEQ ID NO:58=primerMAC1.6f matching sequences in MMAC1 exon 2; SEQ ID NO:59=primer MAC 1.6rmatching sequences in MMAC1 exon 5; SEQ ID NO:60=translation of bases1-54 of SEQ ID NO:56; SEQ ID NO:61=translation of bases 58-96 of SEQ IDNO:56; SEQ ID NO:62=translation of bases 98-178 of SEQ ID NO:56; SEQ IDNO:63=translation of bases 182-208 of SEQ ID NO:56; SEQ IDNO:64=sequence of human TS10q23.3 pseudogene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. The Present Invention

As stated above, a number of different groups have shown evidence of atumor suppressing activity associated with the 10q region of humanchromosome 10. Despite this considerable amount of work, the identity ofthe gene or genes responsible for this activity has not been determined.Previous investigations used a functional approach involving transfer ofchromosomes or chromosomal fragments suspected of harboring tumorsuppressor gene(s) into tumorigenic glioma cells. These efforts alloweddefinition of the biological activity of putative tumor suppressorgene(s) and aided in the localization of such activity. Chromosomes 2and 10 were transferred into U251 glioma cells and chromosomes 2 and 10into LG-11 cells. The LG-11 cells were shown to have no intact copies ofchromosome 10 and the breakpoint was subsequently found to occur at10q24. The transfer of chromosome 10 resulted in hybrid cells thatdisplayed a suppressed phenotype, exhibiting a loss of tumorigenicity(no tumor formation) and loss of the ability to grow in soft agarose(50× to 1000× decrease; Pershouse et al., 1993). The hybrid'sexponential growth rate was similar to the parental cell, although thehybrid cell's saturation density was significantly (10× to 20×) lowerthan the parental cells. The transfer of chromosome 2 resulted in hybridcells that acted similar to the parental cells.

One objective of these studies was to localize the chromosome 10suppressor gene by fragmentation of the neomycin-tagged chromosome 10and, subsequently, to transfer the fragmented chromosome into gliomacells. However, the inventors observed that some of the hybrid cells hadspontaneously undergone chromosomal rearrangements to yield hybrid cellsretaining only various regions of the inserted chromosome 10 (Pershouseet al., 1993). The inventors then subcloned the hybrids and analyzedthem, rather than initiate fragmentation studies (Steck et al., 1995).The retention of the inserted chromosome 10 or its fragments was trackedby informative RFLP markers and FISH analysis. Interestingly, only theinserted chromosome was subjected to rearrangement. The insertion of anentire copy of chromosome 10 resulted in inhibition of the hybrid cell'stransformed property to proliferate in soft agarose and to form tumorsin nude mice.

These two phenotypes now appear to be partially separable by the instantanalysis. Some subclones (U251.N10.5a-j), which revealed a loss of amajor portion of the long arm of chromosome 10, grew in soft agarose butfailed to form tumors in nude mice, thus indicating that a tumorsuppressive locus resides in the remaining portion of the chromosome(10pter to 10q11). In contrast, clones that retained a distal region ofthe long arm, 10q24 to 10q26, failed both to proliferate in soft agaroseand in nude mice (see FIG. 4). This suggests another phenotypicsuppressive region residing in the distal region of the chromosome. Thelack of additional 10-associated material further would suggest that theremaining chromosome 10 material is responsible for the alteredbiological phenotype. These results implicate the presence of twophenotypically independent suppressive regions on chromosome 10 involvedin glioma progression (Steck et al., 1995).

According to the present invention, the inventors now have used severalindependent strategies to localize a tumor suppressor gene, designatedTS10q23.3, that is involved in gliomas, breast cancer, prostate cancerand other cancers. These approaches, described in greater detail in thefollowing Examples, included (i) identification of homozygous deletionsin a series of human glioma cell lines; (ii) determination of aconsistent region(s) of retention in clones suppressed fortumorigenicity; and (iii) allelic deletion studies on various grades ofglioma and corresponding normal samples. With the gene in hand, it nowbecomes possible to exploit the information encoded by the gene todevelop novel diagnostic and therapeutic approaches related to humancancer.

II. The 10q23.3 Tumor Suppressor

According to the present invention, there has been identified a tumorsuppressor, encoded by a gene in the 10q23.3 locus, and designated hereas TS10q23.3. This molecule is capable of suppressing tumor phenotypesin various cancers. The term tumor suppressor is well-known to those ofskill in the art. Examples of other tumors suppressors are p53, Rb andp16, to name a few. While these molecules are structurally distinct,they form a group of functionally-related molecules, of which TS10q23.3is a member. The uses in which these other tumor suppressors now arebeing exploited are equally applicable here.

In addition to the entire TS10q23.3 molecule, the present invention alsorelates to fragments of the polypeptide that may or may not retain thetumor suppressing (or other) activity. Fragments, including theN-terminus of the molecule may be generated by genetic engineering oftranslation stop sites within the coding region (discussed below).Alternatively, treatment of the TS10q23.3 molecule with proteolyticenzymes, known as proteases, can produces a variety of N-terminal,C-terminal and internal fragments. Examples of fragments may includecontiguous residues of the TS10q23.3. sequence given in FIG. 7 and FIG.9, of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100,200, 300, 400 or more amino acids in length. These fragments may bepurified according to known methods, such as precipitation (e.g.,ammonium sulfate), HPLC, ion exchange chromatography, affinitychromatography (including immunoaffinity chromatography) or various sizeseparations (sedimentation, gel electrophoresis, gel filtration).

A. Structural Features of the Polypeptide

The gene for TS10q23.3 encodes a 403 amino acid polypeptide. Thepredicted molecular weight of this molecule is 47,122, with a resultingpI of 5.86. Thus, at a minimum, this molecule may be used as a standardin assays where molecule weight and pI are being examined.

A phosphatase consensus site is located at residues 122-131, matchingperfectly the tyrosine phosphatase (PTP) consensus sequence:[I/V]HCxAGxxR[S/T]G. Outside the active domains, sequences differgreatly. PTPs proceed through phosphoenzyme intermediates. The enzymaticreaction involves phosphoryl-cysteine intermediate formation afternucleophilic attack of the phosphorus atom of the substrate by thethiolate anion of cysteine. The reaction can be represented as atwo-step chemical process: phosphoryl transfer to the enzyme accompaniedby rapid release of dephosphorylated product; and hydrolysis of thethiol-phosphate intermediate concomitant with rapid release ofphosphate. To form the catalytically competent component complex, theenzyme binds and reacts with the dianion of phosphate-containingsubstrate. On the enzyme, an aspartic acid must be protonated and thenucleophilic cysteine must be unprotonated (thiolate anion) forphosphoryl transfer to the enzyme. Also of note are potential tyrosinephosphorylation sites located at residues 233-240 and 308-315 and cAMPphosphorylation sites located at residues 128, 164, 223 and 335.Phosphatases are known to have kinase sites, and the phosphataseactivity of these enzymes can be modulated by phosphorylation at thesesites. Protein phosphatases generally are divided into twocategories—serine/threonine phosphatases and tyrosine phosphatases.Certain of the tyrosine phosphatases also have activity againstphosphoserine and phosphothreonine.

The interaction between kinases and phosphatases, and the variousphosphorylation states of polypeptides, have been demonstrated asimportant features in cell physiology. Through a variety of differentmechanisms, kinases and phosphatases act in different pathways withincells that are involved in signaling, energy storage and cellregulation. Since the identification of an intrinsic tyrosine kinasefunction in the transforming protein src (Collett & Erickson, 1978), therole of phosphorylation, particularly on tyrosine residues, has beendemonstrated to be critical in the control of cellular proliferation andthe induction of cancer (Hunter, 1991; Bishop, 1991). The roles thatprotein phosphatases play in growth regulation, as well as in many otherbiological and biochemical activities, have been correlated with thephosphorylation state of biologically important molecules (Cohen, 1994).

Based on its sequence, TS10q23.3 appears to encode a tyrosinephosphatase or dual specificity phosphatase with homology to thecytoskeleton associated proteins, chicken tensin and bovine auxilin(Steck et al., 1997; Li et al., 1997). The N-terminal half of TS10q23.3is homologous to several phosphatases and its putative core phosphatasemotif is present at residues 122-134 (Denu et al., 1996; Tonks and Neel,1996). Thus, the N-terminal half of TS10q23.3 is homologous to severalphosphatases and its putative core phosphatase motif is present atresidues 122-134 (Denu et al., 1996; Tonks and Neel, 1996). Thus, theN-terminal region of TS10q23.3 may have enzymatic and cellularlocalization activities. The C-terminal portion of TS10q23.3 containsthree potential tyrosine phosphorylation sites at residues 240, 315 and336. If phosphorylated, tyrosine 315 would represent a potential SH2binding site as there is a leucine residue located three residuesC-terminal from the tyrosine (Songyang et al., 1995). Two potentialserine phosphorylation sites are also present within the C-terminal halfof TS10Q23.3. Serine residue 338 represents a potentialCa2+/calmodulin-dependent protein kinase II site, while serine 355represents a potential caseine kinase II site (Hardie and Hanks, 1995).The last four C-terminal amino acids, ITKV, represent a potential PDZbinding domain (Fanning and Anderson, 1996; Saras and Heldin, 1996). PDZdomains are present in a variety of intracellular proteins and arethought to mediate protein-protein interactions by binding directly tothe C-terminal ends of target proteins.

It also should be mentioned that the 60 or so amino acids of theN-terminus of the molecule show some homology to tensin, a cytoskeletalprotein implicated in adhesion plaques. This suggests that TS10q23.3 maybe involved in cell surface phenomena such as contact inhibition,invasion, migration or cell-to-cell signaling. TS10q23.3 point mutationsidentified in certain tumor cell lines affect secondary proposedstructures in this region.

B. Functional Aspects

When the present application refers to the function of TS10q23.3 or“wild-type” activity, it is meant that the molecule in question has theability to inhibit the transformation of a cell from a normallyregulated state of proliferation to a malignant state, i.e., oneassociated with any sort of abnormal growth regulation, or to inhibitthe transformation of a cell from an abnormal state to a highlymalignant state, e.g., to prevent metastasis or invasive tumor growth.Other phenotypes that may be considered to be regulated by the normalTS10q23.3 gene product are angiogenesis, adhesion, migration,cell-to-cell signaling, cell growth, cell proliferation,density-dependent growth, anchorage-dependent growth and others.Determination of which molecules possess this activity may be achievedusing assays familiar to those of skill in the art. For example,transfer of genes encoding TS10q23.3, or variants thereof, into cellsthat do not have a functional TS10q23.3 product, and hence exhibitimpaired growth control, will identify, by virtue of growth suppression,those molecules having TS10q23.3 function.

As stated above, there is an indication that TS10q23.3 is a phosphatase.The portion of the protein located at residues 88 to 98 is a perfectmatch for the conserved catalytic domain of protein tyrosinephosphatase. Also, putative kinase targets are located in the molecule,which is another characteristic of phosphatases. Because other tumorsuppressors have been identified with this type of activity, it will bedesirable to determine the phosphatase function in the tumor suppressingrole of TS10q23.3. This also may be a fruitful approach to developingscreening assays for the absence of TS10q23.3 function or in thedevelopment of cancer therapies, for example, in targeting thephosphatase function of TS10q23.3, targeting the substrate upon whichTS10q23.2 acts, and/or targeting the kinase or kinases which act uponTS10q23.3.

C. Variants of TS10q23.3

Amino acid sequence variants of the polypeptide can be substitutional,insertional or deletion variants. Deletion variants lack one or moreresidues of the native protein which are not essential for function orimmunogenic activity, and are exemplified by the variants lacking atransmembrane sequence described above. Another common type of deletionvariant is one lacking secretory signal sequences or signal sequencesdirecting a protein to bind to a particular part of a cell. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

In particular aspects it is contemplated that one of skill in the artwill employ standard technologies well known to those of skill in theart to produce the mutants. Specifically contemplated will be N-terminaldeletions, C-terminal deletions, internal deletions, as well as randomand point mutagenesis.

N-terminal and C-terminal deletions are forms of deletion mutagenesisthat take advantage for example, of the presence of a suitable singlerestriction site near the end of the C- or N-terminal region. The DNA iscleaved at the site and the cut ends are degraded by nucleases such asBAL31, exonuclease III, DNase I, and S1 nuclease. Rejoining the two endsproduces a series of DNAs with deletions of varying size around therestriction site. Proteins expressed from such mutant can be assayed forapoptosis inhibiting and/or chaperone function as described throughoutthe specification. Similar techniques are employed in internal deletionmutants, however, in internal deletion mutants are generated by usingtwo suitably placed restriction sites, thereby allowing a preciselydefined deletion to be made, and the ends to be religated as above.

Also contemplated are partial digestions mutants. In such instances, oneof skill in the art would employ a “frequent cutter”, that cuts the DNAin numerous places depending on the length of reaction time. Thus, byvarying the reaction conditions it will be possible to generate a seriesof mutants of varying size, which may then be screened for activity.

A random insertional mutation may also be performed by cutting the DNAsequence with a DNase I, for example, and inserting a stretch ofnucleotides that encode, 3, 6, 9, 12 etc., amino acids and religatingthe end. Once such a mutation is made the mutants can be screened forvarious activities presented by the wild-type protein.

Once general areas of the gene are identified as encoding particularprotein domains, point mutagenesis may be employed to identify withparticularity which amino acid residues are important in particularactivities associated with TS10Q23.3. Thus one of skill in the art willbe able to generate single base changes in the DNA strand to result inan altered codon and a missense mutation.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventors thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte & Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. A peptide mimetic is expected topermit molecular interactions similar to the natural molecule. Theseprinciples may be used, in conjunction with the principles outlineabove, to engineer second generation molecules having many of thenatural properties of TS10q23.3, but with altered and even improvedcharacteristics.

D. Domain Switching

As described in the examples, the present inventors have identifiedputative murine and canine homologs of the human TS10q23.3 gene. Inaddition, mutations have been identified in TS10q23.3 which are believedto alter its function. These studies are important for at least tworeasons. First, they provide a reasonable expectation that still otherhomologs, allelic variants and mutants of this gene may exist in relatedspecies, such as rat, rabbit, monkey, gibbon, chimp, ape, baboon, cow,pig, horse, sheep and cat. Upon isolation of these homologs, variantsand mutants, and in conjunction with other analyses, certain active orfunctional domains can be identified. Second, this will provide astarting point for further mutational analysis of the molecule. One wayin which this information can be exploited is in “domain switching.”

Domain switching involves the generation of chimeric molecules usingdifferent but, in this case, related polypeptides. By comparing themouse, dog and human sequences for TS10q23.3 with the TS10q23.3 of otherspecies, and with mutants and allelic variants of these polypeptides,one can make predictions as to the functionally significant regions ofthese molecules. It is possible, then, to switch related domains ofthese molecules in an effort to determine the criticality of theseregions to TS10q23.3 function. These molecules may have additional valuein that these “chimeras” can be distinguished from natural molecules,while possibly providing the same function.

Based on the sequence identity, at the amino acid level, of the mouse,dog and human sequences, it may be inferred that even small changes inthe primary sequence of the molecule will affect function. Furtheranalysis of mutations and their predicted effect on secondary structurewill add to this understanding.

Another structural aspect of TS10q23.3 that provides fertile ground fordomain switching experiments is the tyrosine phosphatase-like domain andthe putative tyrosine phosphorylation sites. This domain may besubstituted for other phosphatase domains in order to alter thespecificity of this function. A further investigation of the homologybetween TS10q23.3 and other phosphatases is warranted by thisobservation.

E. Fusion Proteins

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of a immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

One particular fusion of interest would include a deletion constructlacking the phosphatase site of TS10q23.3 but containing other regionsthat could bind the substrate molecule. Fusion to a polypeptide that canbe used for purification of the substrate-TS10q23.3 complex would serveto isolated the substrate for identification and analysis.

Examples of fusion protein expression systems include the glutathioneS-transferase (GST) system (Pharmacia, Piscataway, N.J.), the maltosebinding protein system (NEB, Beverley, Mass.), the FLAG system (IBI, NewHaven, Conn.), and the 6×His system (Qiagen, Chatsworth, Calif.).

Some of these systems produce recombinant polypeptides bearing only asmall number of additional amino acids, which are unlikely to affect theantigenic ability of the recombinant polypeptide. For example, both theFLAG system and the 6×His system add only short sequences, both of whichare known to be poorly antigenic and which do not adversely affectfolding of the polypeptide to its native conformation.

In still further systems, it is possible to create fusion proteinconstructs to enhance immunogenicity of a TS10q23.3 fusion construct toincrease immunogenicity are well known to those of skill in the art, forexample, a fusion of TS10q23.3 with a helper antigen such as hsp70 orpeptide sequences such as from Diptheria toxin chain or a cytokine suchas IL2 will be useful in eliciting an immune response. In otherembodiments, fusion construct can be made which will enhance thetargeting of the TS10q23.3 related compositions to a specific site orcell. For example, fusing TS10q23.3 or a TS10q23.3 type protein to aligand will be an effective means to target the composition to a siteexpressing the receptor for such a ligand. In this manner the TS10q23.3or TS10q23.3 related composition may be delivered into a cell viareceptor mediated delivery. The TS10q23.3 protein can be attachedcovalently or fused to a ligand. This can be used as a mechanics fordelivery into a cell. The ligand with the protein attached may then beinternalized by a receptor bearing cell.

Other fusion systems produce polypeptide hybrids where it is desirableto excise the fusion partner from the desired polypeptide. In oneembodiment, the fusion partner is linked to the recombinant TS10q23.3polypeptide by a peptide sequence containing a specific recognitionsequence for a protease. Examples of suitable sequences are thoserecognized by the Tobacco Etch Virus protease (Life Technologies,Gaithersburg, Md.) or Factor Xa (New England Biolabs, Beverley, Mass.).

F. Purification of Proteins

It will be desirable to purify TS10q23.3 or variants thereof. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

G. Synthetic Peptides

The present invention also describes smaller TS10q23.3-related peptidesfor use in various embodiments of the present invention. Because oftheir relatively small size, the peptides of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1984); Tam et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979), each incorporated herein byreference. Short peptide sequences, or libraries of overlappingpeptides, usually from about 6 up to about 35 to 50 amino acids, whichcorrespond to the selected regions described herein, can be readilysynthesized and then screened in screening assays designed to identifyreactive peptides. Alternatively, recombinant DNA technology may beemployed wherein a nucleotide sequence which encodes a peptide of theinvention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

U.S. Pat. No. 4,554,101 (incorporated herein by reference) also teachesthe identification and preparation of epitopes from primary amino acidsequences on the basis of hydrophilicity. Through the methods disclosedin Hopp, one of skill in the art would be able to identify epitopes fromwithin any amino acid sequence encoded by any of the DNA sequencesdisclosed herein.

H. Antigen Compositions

The present invention also provides for the use of TS10q23.3 proteins orpeptides as antigens for the immunization of animals relating to theproduction of antibodies. It is envisioned that either TS10q23.3, orportions thereof, will be coupled, bonded, bound, conjugated orchemically-linked to one or more agents via linkers, polylinkers orderivatized amino acids. This may be performed such that a bispecific ormultivalent composition or vaccine is produced. It is further envisionedthat the methods used in the preparation of these compositions will befamiliar to those of skill in the art and should be suitable foradministration to animals, i.e., pharmaceutically acceptable. Preferredagents are the carriers are keyhole limpet hemocyannin (KLH) or bovineserum albumin (BSA).

III. Nucleic Acids

The present invention also provides, in another embodiment, genesencoding TS10q23.3. Genes for the human, canine and murine TS10q23.3molecule have been identified. The present invention is not limited inscope to these genes, however, as one of ordinary skill in the could,using these nucleic acids, readily identify related homologs in variousother species (e.g., rat, rabbit, monkey, gibbon, chimp, ape, baboon,cow, pig, horse, sheep, cat and other species). The finding of mouse anddog homologs for this gene makes it likely that other species moreclosely related to humans will, in fact, have a homolog as well.

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, a “TS10q23.3 gene” may contain a variety of different bases andyet still produce a corresponding polypeptide that is functionallyindistinguishable, and in some cases structurally, from the human andmouse genes disclosed herein.

Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Inaddition to therapeutic considerations, cells expressing nucleic acidsof the present invention may prove useful in the context of screeningfor agents that induce, repress, inhibit, augment, interfere with,block, abrogate, stimulate or enhance the function of TS10q23.3.

A. Nucleic Acids Encoding 10q23.3

The human gene disclosed in FIGS. 6 and 9, and the murine gene disclosedin FIG. 9 are TS10q23.3 genes of the present invention. Nucleic acidsaccording to the present invention may encode an entire TS10q23.3 gene,a domain of TS10q23.3 that expresses a tumor suppressing or phosphatasefunction, or any other fragment of the TS10q23.3 sequences set forthherein. The nucleic acid may be derived from genomic DNA, i.e., cloneddirectly from the genome of a particular organism. In preferredembodiments, however, the nucleic acid would comprise complementary DNA(cDNA). Also contemplated is a cDNA plus a natural intron or an intronderived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and other nucleic acidsof the present invention may be used as molecular weight standards in,for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a given TS10q23.3 from a given species maybe represented by natural variants that have slightly different nucleicacid sequences but, nonetheless, encode the same protein (see Table 1below).

As used in this application, the term “a nucleic acid encoding aTS10q23.3” refers to a nucleic acid molecule that has been isolated freeof total cellular nucleic acid. In preferred embodiments, the inventionconcerns a nucleic acid sequence essentially as set forth in FIGS. 6 and9. The term “as set forth in FIG. 6 or 9” means that the nucleic acidsequence substantially corresponds to a portion of FIG. 6 or 9. The term“functionally equivalent codon” is used herein to refer to codons thatencode the same amino acid, such as the six codons for arginine orserine (Table 1, below), and also refers to codons that encodebiologically equivalent amino acids, as discussed in the followingpages. TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU CysteineCys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of FIG. 9 will be sequences that are “as set forth in FIG.9.” Sequences that are essentially the same as those set forth in FIG. 9may also be functionally defined as sequences that are capable ofhybridizing to a nucleic acid segment containing the complement of FIG.9 under standard conditions.

The DNA segments of the present invention include those encodingbiologically functional equivalent TS10q23.3 proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

B. Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin FIGS. 6 and 9. Nucleic acid sequences that are “complementary” arethose that are capable of base-pairing according to the standardWatson-Crick complementary rules. As used herein, the term“complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of FIGS. 6 and 9 underrelatively stringent conditions such as those described herein. Suchsequences may encode the entire TS10q23.3 protein or functional ornon-functional fragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used,although others are contemplated. Longer polynucleotides encoding 250,500, 1000, 1212, 1500, 2000, 2500, 3000 or 3431 bases and longer arecontemplated as well. Such oligonucleotides will find use, for example,as probes in Southern and Northern blots and as primers in amplificationreactions.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

One method of using probes and primers of the present invention is inthe search for genes related to TS10q23.3 or, more particularly,homologs of TS10q23.3 from other species. The existence of a murinehomolog strongly suggests that other homologs of the human TS10q23.3will be discovered in species more closely related, and perhaps moreremote, than mouse. Normally, the target DNA will be a genomic or cDNAlibrary, although screening may involve analysis of RNA molecules. Byvarying the stringency of hybridization, and the region of the probe,different degrees of homology may be discovered.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

C. Antisense Constructs

In some cases, mutant tumor suppressors may not be non-functional.Rather, they may have aberrant functions that cannot be overcome byreplacement gene therapy, even where the “wild-type” molecule isexpressed in amounts in excess of the mutant polypeptide. Antisensetreatments are one way of addressing this situation. Antisensetechnology also may be used to “knock-out” function of TS10q23.3 in thedevelopment of cell lines or transgenic mice for research, diagnosticand screening purposes.

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with “complementary” sequences. By complementary, it ismeant that polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementarity rules. That is,the larger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

D. Ribozymes

Another approach for addressing the “dominant negative” mutant tumorsuppressor is through the use of ribozymes. Although proteinstraditionally have been used for catalysis of nucleic acids, anotherclass of macromolecules has emerged as useful in this endeavor.Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cook, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cook et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Cook et al., 1981). For example, U.S. Pat. No. 5,354,855 reports thatcertain ribozymes can act as endonucleases with a sequence specificitygreater than that of known ribonucleases and approaching that of the DNArestriction enzymes. Thus, sequence-specific ribozyme-mediatedinhibition of gene expression may be particularly suited to therapeuticapplications (Scanlon et al., 1991; Sarver et al., 1990). Recently, itwas reported that ribozymes elicited genetic changes in some cells linesto which they were applied; the altered genes included the oncogenesH-ras, c-fos and genes of HIV. Most of this work involved themodification of a target mRNA, based on a specific mutant codon that iscleaved by a specific ribozyme.

E. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments expression vectors are employed to expressthe TS10q23.3 polypeptide product, which can then be purified and, forexample, be used to vaccinate animals to generate antisera or monoclonalantibody with which further studies may be conducted. In otherembodiments, the expression vectors are used in gene therapy. Expressionrequires that appropriate signals be provided in the vectors, and whichinclude various regulatory elements, such as enhancers/promoters fromboth viral and mammalian sources that drive expression of the genes ofinterest in host cells. Elements designed to optimize messenger RNAstability and translatability in host cells also are defined. Theconditions for the use of a number of dominant drug selection markersfor establishing permanent, stable cell clones expressing the productsare also provided, as is an element that links expression of the drugselection markers to expression of the polypeptide.

(i) Regulatory Elements

Promoters. Throughout this application, the term “expression construct”is meant to include any type of genetic construct containing a nucleicacid coding for gene products in which part or all of the nucleic acidencoding sequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding genes of interest.

The nucleic acid encoding a gene product is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the synthetic machinery of the cell, or introduced syntheticmachinery, required to initiate the specific transcription of a gene.The phrase “under transcriptional control” means that the promoter is inthe correct location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. The system is based onthe heterodimeric ecdysone receptor of Drosophila, and when ecdysone oran analog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constitutively expressed fromone vector, whereas the ecdysone-responsive promoter which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.) originally developed by Gossen andBujard (Gossen and Bujard, 1992; Gossen et al., 1995). This system alsoallows high levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On™ system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off™ system, gene expression is turnedon in the absence of doxycycline. These systems are based on tworegulatory elements derived from the tetracycline resistance operon ofE. coli. The tetracycline operator sequence to which the tetracyclinerepressor binds, and the tetracycline repressor protein. The gene ofinterest is cloned into a plasmid behind a promoter that hastetracycline-responsive elements present in it. A second plasmidcontains a regulatory element called the tetracycline-controlledtransactivator, which is composed, in the Tet-Off™ system, of the VP16domain from the herpes simplex virus and the wild-type tertracyclinerepressor. Thus in the absence of doxycycline, transcription isconstitutively on. In the Tet-On™ system, the tetracycline repressor isnot wild type and in the presence of doxycycline activatestranscription. For gene therapy vector production, the Tet-Off™ systemwould be preferable so that the producer cells could be grown in thepresence of tetracycline or doxycycline and prevent expression of apotentially toxic transgene, but when the vector is introduced to thepatient, the gene expression would be constituitively on.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters may be used to effect transcriptionin specific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate. Similarly, the following promoters may be used to target geneexpression in other tissues (Table 2). TABLE 2 Tissue specific promotersTissue Promoter Pancreas insulin elastin amylase pdr-1 pdx-1 glucokinaseLiver albumin PEPCK HBV enhancer alpha fetoprotein apolipoprotein Calpha-1 antitrypsin vitellogenin, NF-AB Transthyretin Skeletal musclemyosin H chain muscle creatine kinase dystrophin calpain p94 skeletalalpha-actin fast troponin 1 Skin keratin K6 keratin K1 Lung CFTR humancytokeratin 18 (K18) pulmonary surfactant proteins A, B and C CC-10 P1Smooth muscle sm22 alpha SM-alpha-actin Endothelium endothelin-1E-selectin von Willebrand factor TIE (Korhonen et al., 1995) KDR/flk-1Melanocytes tyrosinase Adipose tissue lipoprotein lipase (Zechner etal., 1988) adipsin (Spiegelman et al., 1989) acetyl-CoA carboxylase(Pape and Kim, 1989) glycerophosphate dehydrogenase (Dani et al., 1989)adipocyte P2 (Hunt et al., 1986) Blood β-globin

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. For example in gene therapy applications where theindication is a gonadal tissue where specific steroids are produced orrouted to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV,MT-1, ecdysone and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary and adrenal hormones are expectedto be useful in the present invention. Cytokine and inflammatory proteinresponsive promoters that could be used include K and T Kininogen(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone etal., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBPalpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson etal., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988),alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988),angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible byphorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin and alpha-1 antichymotrypsin.

It is envisioned that cell cycle regulatable promoters may be useful inthe present invention. For example, in a bi-cistronic gene therapyvector, use of a strong CMV promoter to drive expression of a first genesuch as p16 that arrests cells in the G1 phase could be followed byexpression of a second gene such as p53 under the control of a promoterthat is active in the G1 phase of the cell cycle, thus providing a“second hit” that would push the cell into apoptosis. Other promoterssuch as those of various cyclins, PCNA, galectin-3, E2F1, p53 and BRCA1could be used.

Tumor specific promoters such as osteocalcin, hypoxia-responsive element(HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may alsobe used to regulate gene expression in tumor cells. Other promoters thatcould be used according to the present invention includeLac-regulatable, chemotherapy inducible (e.g. MDR), and heat(hyperthermia) inducible promoters, radiation-inducible (e.g., EGR (Jokiet al., 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acidpromoters, U1 snRNA (Bartlett et al., 1996), MC-1, PGK, β-actin andα-globin. Many other promoters that may be useful are listed in Waltherand Stein (1996).

It is envisioned that any of the above promoters alone or in combinationwith another may be useful according to the present invention dependingon the action desired. In addition, this list of promoters is should notbe construed to be exhaustive or limiting, those of skill in the artwill know of other promoters that may be used in conjunction with thepromoters and methods disclosed herein.

Enhancers. Enhancers are genetic elements that increase transcriptionfrom a promoter located at a distant position on the same molecule ofDNA. Enhancers are organized much like promoters. That is, they arecomposed of many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Below is a list of promoters additional to the tissue specific promoterslisted above, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the nucleicacid encoding a gene of interest in an expression construct (Table 3 andTable 4). Additionally, any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct. TABLE 3ENHANCER/PROMOTER Immunoglobulin Heavy Chain Immunoglobulin Light ChainT-Cell Receptor HLA DQ α and DQ β β-Interferon Interleukin-2Interleukin-2 Receptor MHC Class II 5 MHC Class II HLA-DRα β-ActinMuscle Creatine Kinase Prealbumin (Transthyretin) Elastase IMetallothionein Collagenase Albumin Gene α-Fetoprotein τ-Globin β-Globine-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM)α1-Antitrypsin H2B (TH2B) Histone Mouse or Type I CollagenGlucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone HumanSerum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth FactorDuchenne Muscular Dystrophy SV40 Polyoma Retroviruses Papilloma VirusHepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus GibbonApe Leukemia Virus

TABLE 4 Element Inducer MT II Phorbol Ester (TPA) Heavy metals MMTV(mouse mammary tumor Glucocorticoids virus) β-Interferon poly(rI)Xpoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 PhorbolEster (TPA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kB Interferon HSP70 Ela, SV40 Large T Antigen Proliferin PhorbolEster-TPA Tumor Necrosis Factor FMA Thyroid Stimulating Hormone αThyroid Hormone Gene Insulin E Box Glucose

Polyadenylation Signals. Where a cDNA insert is employed, one willtypically desire to include a polyadenylation signal to effect properpolyadenylation of the gene transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and any such sequence may be employed such ashuman or bovine growth hormone and SV40 polyadenylation signals. Alsocontemplated as an element of the expression cassette is a terminator.These elements can serve to enhance message levels and to minimize readthrough from the cassette into other sequences.

IRES. In certain embodiments of the invention, the use of internalribosome entry site (IRES) elements is contemplated to create multigene,or polycistronic, messages. IRES elements are able to bypass theribosome scanning model of 5′ methylated Cap dependent translation andbegin translation at internal sites (Pelletier and Sonenberg, 1988).IRES elements from two members of the picornavirus family (poliovirusand encephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

(ii) Selectable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstructs of the present invention, a cell may be identified in vitroor in vivo by including a marker in the expression construct. Suchmarkers would confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

(iii) Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kb of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986). The vector may be capable ofreplicating inside the cells. Alternatively, the vector may bereplication deficient and is replicated in helper cells prior todelivery. Suitable vectors are known, such as disclosed in U.S. Pat. No.5,252,479 and PCT published application WO 93/07282 and U.S. Pat. Nos.5,691,198; 5,747,469; 5,436,146 and 5,753,500.

Adenoviruses. One of the preferred methods for in vivo delivery involvesthe use of an adenovirus expression vector. “Adenovirus expressionvector” is meant to include those constructs containing adenovirussequences sufficient to (a) support packaging of the construct and (b)to express an antisense polynucleotide that has been cloned therein. Inthis context, expression does not require that the gene product besynthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5′-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al.,1977). Since the E3 region is dispensable from the adenovirus genome(Jones and Shenk, 1978), the current adenovirus vectors, with the helpof 293 cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Recently, Racher et al., (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al., (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10⁹-10¹¹ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

Retroviruses. The retroviruses are a group of single-stranded RNAviruses characterized by an ability to convert their RNA todouble-stranded DNA in infected cells by a process ofreverse-transcription (Coffin, 1990). The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

Herpesvirus. Because herpes simplex virus (HSV) is neurotropic, it hasgenerated considerable interest in treating nervous system disorders.Moreover, the ability of HSV to establish latent infections innon-dividing neuronal cells without integrating in to the host cellchromosome or otherwise altering the host cell's metabolism, along withthe existence of a promoter that is active during latency makes HSV anattractive vector. And though much attention has focused on theneurotropic applications of HSV, this vector also can be exploited forother tissues given its wide host range.

Another factor that makes HSV an attractive vector is the size andorganization of the genome. Because HSV is large, incorporation ofmultiple genes or expression cassettes is less problematic than in othersmaller viral systems. In addition, the availability of different viralcontrol sequences with varying performance (temporal, strength, etc.)makes it possible to control expression to a greater extent than inother systems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations.

HSV also is relatively easy to manipulate and can be grown to hightiters. Thus, delivery is less of a problem, both in terms of volumesneeded to attain sufficient MOI and in a lessened need for repeatdosings. For a review of HSV as a gene therapy vector, see Glorioso etal. (1995).

HSV, designated with subtypes 1 and 2, are enveloped viruses that areamong the most common infectious agents encountered by humans, infectingmillions of human subjects worldwide. The large, complex,double-stranded DNA genome encodes for dozens of different geneproducts, some of which derive from spliced transcripts. In addition tovirion and envelope structural components, the virus encodes numerousother proteins including a protease, a ribonucleotides reductase, a DNApolymerase, a ssDNA binding protein, a helicase/primase, a DNA dependentATPase, a dUTPase and others.

HSV genes form several groups whose expression is coordinately regulatedand sequentially ordered in a cascade fashion (Honess and Roizman, 1974;Honess and Roizman 1975; Roizman and Sears, 1995). The expression of agenes, the first set of genes to be expressed after infection, isenhanced by the virion protein number 16, or α-transducing factor (Postet al., 1981; Batterson and Roizman, 1983; Campbell et al., 1983). Theexpression of β genes requires functional a gene products, most notablyICP4, which is encoded by the α4 gene (DeLuca et al., 1985). γ genes, aheterogeneous group of genes encoding largely virion structuralproteins, require the onset of viral DNA synthesis for optimalexpression (Holland et al., 1980).

In line with the complexity of the genome, the life cycle of HSV isquite involved. In addition to the lytic cycle, which results insynthesis of virus particles and, eventually, cell death, the virus hasthe capability to enter a latent state in which the genome is maintainedin neural ganglia until some as of yet undefined signal triggers arecurrence of the lytic cycle. Avirulent variants of HSV have beendeveloped and are readily available for use in gene therapy contexts(U.S. Pat. No. 5,672,344).

Adeno-Associated Virus. Recently, adeno-associated virus (AAV) hasemerged as a potential alternative to the more commonly used retroviraland adenoviral vectors. While studies with retroviral and adenoviralmediated gene transfer raise concerns over potential oncogenicproperties of the former, and immunogenic problems associated with thelatter, AAV has not been associated with any such pathologicalindications.

In addition, AAV possesses several unique features that make it moredesirable than the other vectors. Unlike retroviruses, AAV can infectnon-dividing cells; wild-type AAV has been characterized by integration,in a site-specific manner, into chromosome 19 of human cells (Kotin andBerns, 1989; Kotin et al., 1990; Kotin et al., 1991; Samulski et al.,1991); and AAV also possesses anti-oncogenic properties (Ostrove et al.,1981; Berns and Giraud, 1996). Recombinant AAV genomes are constructedby molecularly cloning DNA sequences of interest between the AAV ITRs,eliminating the entire coding sequences of the wild-type AAV genome. TheAAV vectors thus produced lack any of the coding sequences of wild-typeAAV, yet retain the property of stable chromosomal integration andexpression of the recombinant genes upon transduction both in vitro andin vivo (Berns, 1990; Berns and Bohensky, 1987; Bertran et al., 1996;Kearns et al., 1996; Ponnazhagan et al., 1997a). Until recently, AAV wasbelieved to infect almost all cell types, and even cross speciesbarriers. However, it now has been determined that AAV infection isreceptor-mediated (Ponnazhagan et al., 1996; Mizukami et al., 1996).

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.Inverted terminal repeats flank the genome. Two genes are present withinthe genome, giving rise to a number of distinct gene products. Thefirst, the cap gene, produces three different virion proteins (VP),designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes fournon-structural proteins (NS). One or more of these rep gene products isresponsible for transactivating AAV transcription. The sequence of AAVis provided by Srivastava et al. (1983), and in U.S. Pat. No. 5,252,479(entire text of which is specifically incorporated herein by reference).

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, p19 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced. The splice site,derived from map units 42-46, is the same for each transcript. The fournon-structural proteins apparently are derived from the longer of thetranscripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

Vaccinia Virus. Vaccinia virus vectors have been used extensivelybecause of the ease of their construction, relatively high levels ofexpression obtained, wide host range and large capacity for carryingDNA. Vaccinia contains a linear, double-stranded DNA genome of about 186kb that exhibits a marked “A-T” preference. Inverted terminal repeats ofabout 10.5 kb flank the genome. The majority of essential genes appearto map within the central region, which is most highly conserved amongpoxviruses. Estimated open reading frames in vaccinia virus number from150 to 200. Although both strands are coding, extensive overlap ofreading frames is not common.

At least 25 kb can be inserted into the vaccinia virus genome (Smith andMoss, 1983). Prototypical vaccinia vectors contain transgenes insertedinto the viral thymidine kinase gene via homologous recombination.Vectors are selected on the basis of a tk-phenotype. Inclusion of theuntranslated leader sequence of encephalomyocarditis virus, the level ofexpression is higher than that of conventional vectors, with thetransgenes accumulating at 10% or more of the infected cell's protein in24 h (Elroy-Stein et al., 1989).

Non-Viral transfer. In order to effect expression of sense or antisensegene constructs, the expression construct must be delivered into a cell.This delivery may be accomplished in vitro, as in laboratory proceduresfor transforming cells lines, or in vivo or ex vivo, as in the treatmentof certain disease states. One mechanism for delivery is via viralinfection where the expression construct is encapsidated in aninfectious viral particle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al.,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment of the invention for transferring a nakedDNA expression construct into cells may involve particle bombardment.This method depends on the ability to accelerate DNA-coatedmicroprojectiles to a high velocity allowing them to pierce cellmembranes and enter cells without killing them (Klein et al., 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., 1990). The microprojectiles used have consisted of biologicallyinert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,i.e., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al., (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type suchas lung, epithelial or tumor cells, by any number of receptor-ligandsystems with or without liposomes. For example, epidermal growth factor(EGF) may be used as the receptor for mediated delivery of a nucleicacid encoding a gene in many tumor cells that exhibit upregulation ofEGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

In certain embodiments, gene transfer may more easily be performed underex vivo conditions. Ex vivo gene therapy refers to the isolation ofcells from an animal, the delivery of a nucleic acid into the cells invitro, and then the return of the modified cells back into an animal.This may involve the surgical removal of tissue/organs from an animal orthe primary culture of cells and tissues.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt-cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent T-cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use—the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon. Cells are grown ina stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1.The culture is usually mixed with one or more agitators, based on bladeddisks or marine propeller patterns. Agitator systems offering less shearforces than blades have been described. Agitation may be driven eitherdirectly or indirectly by magnetically coupled drives. Indirect drivesreduce the risk of microbial contamination through seals on stirrershafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gases and generatesrelatively low shear forces.

The antibodies of the present invention are particularly useful for theisolation of antigens by immunoprecipitation. Immunoprecipitationinvolves the separation of the target antigen component from a complexmixture, and is used to discriminate or isolate minute amounts ofprotein. For the isolation of membrane proteins cells must besolubilized into detergent micelles. Nonionic salts are preferred, sinceother agents such as bile salts, precipitate at acid pH or in thepresence of bivalent cations. Antibodies are and their uses arediscussed further, below.

III. Generating Antibodies Reactive with TS10q23.3

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a TS10q23.3 molecule of the present invention, orany portion thereof. An antibody can be a polyclonal or a monoclonalantibody. In a preferred embodiment, an antibody is a monoclonalantibody. Means for preparing and characterizing antibodies are wellknown in the art (see, e.g., Howell and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a non-human animal includingrabbits, mice, rats, hamsters, pigs or horses. Because of the relativelylarge blood volume of rabbits, a rabbit is a preferred choice forproduction of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to TS10q23.3-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular TS10q23.3 of different species may be utilized in otheruseful applications.

In general, both polyclonal and monoclonal antibodies against TS10q23.3may be used in a variety of embodiments. For example, they may beemployed in antibody cloning protocols to obtain cDNAs or genes encodingother TS10q23.3. They may also be used in inhibition studies to analyzethe effects of TS10q23.3 related peptides in cells or animals.Anti-TS10q23.3 antibodies will also be useful in immunolocalizationstudies to analyze the distribution of TS10q23.3 during various cellularevents, for example, to determine the cellular or tissue-specificdistribution of TS10q23.3 polypeptides under different points in thecell cycle. A particularly useful application of such antibodies is inpurifying native or recombinant TS10q23.3, for example, using anantibody affinity column. The operation of all such immunologicaltechniques will be known to those of skill in the art in light of thepresent disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified TS10q23.3 protein, polypeptide or peptide or cellexpressing high levels of TS10q23.3. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bu1; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withcell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

The individual cell lines could also be cultured in vitro, where theMAbs are naturally secreted into the culture medium from which they canbe readily obtained in high concentrations.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the purified monoclonal antibodiesby methods which include digestion with enzymes, such as pepsin orpapain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It also is contemplated that a molecular cloning approach may be used togenerate monoclonals. For this, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and control cellse.g., normal-versus-tumor cells. The advantages of this approach overconventional hybridoma techniques are that approximately 10⁴ times asmany antibodies can be produced and screened in a single round, and thatnew specificities are generated by H and L chain combination whichfurther increases the chance of finding appropriate antibodies.

Humanized monoclonal antibodies are antibodies of animal origin thathave been modified using genetic engineering techniques to replaceconstant region and/or variable region framework sequences with humansequences, while retaining the original antigen specificity. Suchantibodies are commonly derived from rodent antibodies with specificityagainst human antigens. such antibodies are generally useful for in vivotherapeutic applications. This strategy reduces the host response to theforeign antibody and allows selection of the human effector functions.

The techniques for producing humanized immunoglobulins are well known tothose of skill in the art. For example U.S. Pat. No. 5,693,762 disclosesmethods for producing, and compositions of, humanized immunoglobulinshaving one or more complementarity determining regions (CDR's). Whencombined into an intact antibody, the humanized immunoglobulins aresubstantially non-immunogenic in humans and retain substantially thesame affinity as the donor immunoglobulin to the antigen, such as aprotein or other compound containing an epitope.

Other U.S. patents, each incorporated herein by reference, that teachthe production of antibodies useful in the present invention includeU.S. Pat. No. 5,565,332, which describes the production of chimericantibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 whichdescribes recombinant immunoglobin preparations and U.S. Pat. No.4,867,973 which describes antibody-therapeutic agent conjugates.

U.S. Pat. No. 5,565,332 describes methods for the production ofantibodies, or antibody fragments, which have the same bindingspecificity as a parent antibody but which have increased humancharacteristics. Humanized antibodies may be obtained by chainshuffling, perhaps using phage display technology, in as much as suchmethods will be useful in the present invention the entire text of U.S.Pat. No. 5,565,332 is incorporated herein by reference. Human antibodiesmay also be produced by transforming B cells with EBV and subsequentcloning of secretors as described by Hoon et al., (1993).

Antibody conjugates in which a TS10Q23.3 antibody is linked to adetectable label or a cytotoxic agent form further aspects of theinvention. Diagnostic antibody conjugates may be used both in vitrodiagnostics, as in a variety of immunoassays, and in vivo diagnostics,such as in imaging technology.

Certain antibody conjugates include those intended primarily for use invitro, where the antibody is linked to a secondary binding ligand or toan enzyme (an enzyme tag) that will generate a colored product uponcontact with a chromogenic substrate. Examples of suitable enzymesinclude urease, alkaline phosphatase, (horseradish) hydrogen peroxidaseand glucose oxidase. Preferred secondary binding ligands are biotin andavidin or streptavidin compounds. The use of such labels is well knownto those of skill in the art in light and is described, for example, inU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149 and 4,366,241; each incorporated herein by reference.

Radioactively labeled monoclonal antibodies of the present invention maybe produced according to well-known methods in the art. For instance,monoclonal antibodies can be iodinated by contact with sodium orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Monoclonal antibodies according to the invention may be labeled withtechnetium-^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody.

Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to antibody arediethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetraceticacid (EDTA). Fluorescent labels include rhodamine, fluoresceinisothiocyanate and renographin.

IV. Diagnosing Cancers Involving TS10q23.3

The present inventors have determined that alterations in TS10q23.3 areassociated with malignancy. Therefore, TS10q23.3 and the correspondinggene may be employed as a diagnostic or prognostic indicator of cancer.More specifically, point mutations, deletions, insertions or regulatorypertubations relating to TS10q23.3 may cause cancer or promote cancerdevelopment, cause or promoter tumor progression at a primary site,and/or cause or promote metastasis. Other phenomena associated withmalignancy that may be affected by TS10q23.3 expression includeangiogenesis and tissue invasion.

A. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingvariation in the expression of TS10q23.3. This may comprises determiningthat level of TS10q23.3 or determining specific alterations in theexpressed product. Obviously, this sort of assay has importance in thediagnosis of related cancers. Such cancer may involve cancers of thebrain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma,ependymomas), lung, liver, spleen, kidney, pancreas, small intestine,blood cells, lymph node, colon, breast, endometrium, stomach, prostate,testicle, ovary, skin, head and neck, esophagus, bone marrow, blood orother tissue. In particular, the present invention relates to thediagnosis of gliomas.

The biological sample can be any tissue or fluid. Various embodimentsinclude cells of the skin, muscle, facia, brain, prostate, breast,endometrium, lung, head & neck, pancreas, small intestine, blood cells,liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymphnode, bone marrow or kidney. Other embodiments include fluid samplessuch as peripheral blood, lymph fluid, ascites, serous fluid, pleuraleffusion, sputum, cerebrospinal fluid, lacrimal fluid, stool or urine.

Nucleic acid used is isolated from cells contained in the biologicalsample, according to standard methodologies (Sambrook et al., 1989). Thenucleic acid may be genomic DNA or fractionated or whole cell RNA. WhereRNA is used, it may be desired to convert the RNA to a complementaryDNA. In one embodiment, the RNA is whole cell RNA; in another, it ispoly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given patientwith a statistically significant reference group of normal patients andpatients that have TS10q23.3-related pathologies. In this way, it ispossible to correlate the amount or kind of TS10q23.3 detected withvarious clinical states.

Various types of defects have been identified by the present inventors.Thus, “alterations” should be read as including deletions, insertions,point mutations and duplications. Point mutations result in stop codons,frameshift mutations or amino acid substitutions. Somatic mutations arethose occurring in non-germline tissues. Germ-line tissue can occur inany tissue and are inherited. Mutations in and outside the coding regionalso may affect the amount of TS10q23.3 produced, both by altering thetranscription of the gene or in destabilizing or otherwise altering theprocessing of either the transcript (mRNA) or protein.

A cell takes a genetic step toward oncogenic transformation when oneallele of a tumor suppressor gene is inactivated due to inheritance of agermline lesion or acquisition of a somatic mutation. The inactivationof the other allele of the gene usually involves a somatic micromutationor chromosomal allelic deletion that results in loss of heterozygosity(LOH). Alternatively, both copies of a tumor suppressor gene may be lostby homozygous deletion.

The inventors' initial steps toward identifying new mutations inTS10q23.3 were to prescreen primary tumors and tumor cell lines (TCLs)for LOH within this region of 10q23. Primary tumor specimens and TCLswere examined for LOH using polymorphic short tandem repeat markers onchromosome 10 located near the TS10q23.3 locus (Table 6). In this panelof samples, the inventors observed LOH in primary tumor specimens atfrequencies ranging from 20% in colon specimens to 75% in glioblastomamultiforms (GBMs), with an overall LOH frequency of ˜49%. For TCLs withsample sizes greater than nine, the incidence of LOH varied from 28%(colon) to 82% (GBMs), with an overall frequency of 46%.

In primary tumors exhibiting LOH surrounding the TS10q23.3 locus, theinventors detected a frameshift mutation in breast carcinoma, a nonsensemutation in pediatric GBM, a splicing variant in pediatric GBM and amissense variant in melanoma (Table 7). The inventors also investigatedTCLs exhibited LOH, and identified ten homozygous deletions thataffected the coding regions of TS10q23.3 (FIG. 13A and FIG. 13B). Thehomozygous deletions were present in TCLs from astrocytomas, bladdercarcinoma, breast carcinoma, glioblastoma, lung carcinoma, melanoma, andprostate carcinoma. Whereas two of the cell lines had lost all nineTS10q23.3 exons, the other eight TCLs had homozygously deleted differentcoding portions of the gene. Analysis of the remaining TCLs revealed oneframeshift, one nonsense and seven non-conservative missense variants(Table 7). These particular mutations may be targeted witholigonucleotides specifically designed to identify these mutations, orwith antibodies that distinguish these markers from wild-type TS10q23.3.

It is contemplated that other mutations in the TS10q23.3 gene may beidentified in accordance with the present invention. A variety ofdifferent assays are contemplated in this regard, including but notlimited to, fluorescent in situ hybridization (FISH; U.S. Pat. No.5,633,365 and U.S. Pat. No. 5,665,549, each incorporated herein byreference), direct DNA sequencing, PFGE analysis, Southern or Northernblotting, single-stranded conformation analysis (SSCA), RNAse protectionassay, allele-specific oligonucleotide (ASO), dot blot analysis,denaturing gradient gel electrophoresis (e.g., U.S. Pat. No. 5,190,856incorporated herein by reference), RFLP (e.g., U.S. Pat. No. 5,324,631incorporated herein by reference) and PCR™-SSCP.

(i) Primers and Probes

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences can beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred. Probes are defineddifferently, although they may act as primers. Probes, while perhapscapable of priming, are designed to binding to the target DNA or RNA andneed not be used in an amplification process.

In preferred embodiments, the probes or primers are labeled withradioactive species (³²P, ¹⁴C, ³⁵S, ³H, or other label), with afluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).

(ii) Template Dependent Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety.

Briefly, in PCR™, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR™ amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPO No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention, Walker et al. (1992a). StrandDisplacement Amplification (SDA) is another method of carrying outisothermal amplification of nucleic acids which involves multiple roundsof strand displacement and synthesis, i.e., nick translation. See, U.S.Pat. Nos. 5,270,184 and 5,455,166 and Walker et al. (1992b) for SDA andSpargo et al. (1996) for thermophilic SDA.

Repair Chain Reaction (RCR), involves annealing several probesthroughout a region targeted for amplification, followed by a repairreaction in which only two of the four bases are present. The other twobases can be added as biotinylated derivatives for easy detection. Asimilar approach is used in SDA. Target specific sequences can also bedetected using a cyclic probe reaction (CPR). In CPR, a probe having 3′and 5′ sequences of non-specific DNA and a middle sequence of specificRNA is hybridized to DNA that is present in a sample. Uponhybridization, the reaction is treated with RNase H, and the products ofthe probe identified as distinctive products that are released afterdigestion. The original template is annealed to another cycling probeand the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). See also, U.S. Pat. No. 5,409,818, Fahy et al. (1991) andCompton (1991) for 3SR and NASBA. In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer which has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by an RNA polymerase such as T7 or SP6. Inan isothermal cyclic reaction, the RNA's are reverse transcribed intosingle stranded DNA, which is then converted to double stranded DNA, andthen transcribed once again with an RNA polymerase such as T7 or SP6.The resulting products, whether truncated or complete, indicate targetspecific sequences.

Davey et al., EPO No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase 1), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR™” (Frohman, 1990; Ohara et al., 1989; each herein incorporated byreference in their entirety).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal., (1989), incorporated herein by reference in its entirety.

(iii) Southern/Northern Blotting

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by “blotting” on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

(iv) Separation Methods

It normally is desirable, at one stage or another, to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (Freifelder, 1982).

(v) Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols. See Sambrook et al., 1989.For example, chromophore or radiolabel probes or primers identify thetarget during or following amplification.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the TS10q23.3 gene that may then be analyzed bydirect sequencing.

(vi) Kit Components

All the essential materials and reagents required for detecting andsequencing TS10q23.3 and variants thereof may be assembled together in akit. This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™ etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

(vii) Design and Theoretical Considerations for Relative QuantitativeRT-PCR™

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR™ (RT-PCR™) can be used to determine the relativeconcentrations of specific mRNA species isolated from patients. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

In PCR™, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes limiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCR™amplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR™ reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or cells,the relative abundances of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orcells. This direct proportionality between the concentration of the PCR™products and the relative mRNA abundances is only true in the linearrange of the PCR™ reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR™ for acollection of RNA populations is that the concentrations of theamplified PCR™ products must be sampled when the PCR™ reactions are inthe linear portion of their curves.

The second condition that must be met for an RT-PCR™ experiment tosuccessfully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCR™experiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample. Inthe experiments described below, mRNAs for β-actin, asparaginesynthetase and lipocortin II were used as external and internalstandards to which the relative abundance of other mRNAs are compared.

Most protocols for competitive PCR™ utilize internal PCR™ standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR™ amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCR™assay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR™ is performed as a relative quantitative RT-PCR™with an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5-100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR™ assay with an external standard protocol. Theseassays sample the PCR™ products in the linear portion of theiramplification curves. The number of PCR™ cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR™ assays can be superior to those derived from the relativequantitative RT-PCR™ assay with an internal standard.

One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR™ product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR™ product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

(viii) Chip Technologies

Specifically contemplated by the present inventors are chip-based DNAtechnologies such as those described by Hacia et al. (1996) andShoemaker et al. (1996). Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al. (1994); Fodor et al. (1991).

B. Immunodiagnosis

Antibodies of the present invention can be used in characterizing theTS10q23.3 content of healthy and diseased tissues, through techniquessuch as ELISAs and Western blotting. This may provide a screen for thepresence or absence of malignancy or as a predictor of future cancer.

The use of antibodies of the present invention, in an ELISA assay iscontemplated. For example, anti-TS10q23.3 antibodies are immobilizedonto a selected surface, preferably a surface exhibiting a proteinaffinity such as the wells of a polystyrene microtiter plate. Afterwashing to remove incompletely adsorbed material, it is desirable tobind or coat the assay plate wells with a non-specific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of powdered milk.This allows for blocking of non-specific adsorption sites on theimmobilizing surface and thus reduces the background caused bynon-specific binding of antigen onto the surface.

After binding of antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the sample to be tested in amanner conducive to immune complex (antigen/antibody) formation.

Following formation of specific immunocomplexes between the test sampleand the bound antibody, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for TS10q23.3 that differs thefirst antibody. Appropriate conditions preferably include diluting thesample with diluents such as BSA, bovine gamma globulin (BGG) andphosphate buffered saline (PBS)/Tween®. These added agents also tend toassist in the reduction of nonspecific background. The layered antiserais then allowed to incubate for from about 2 to about 4 hr, attemperatures preferably on the order of about 25° to about 27° C.Following incubation, the antisera-contacted surface is washed so as toremove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween®, or borate buffer.

To provide a detecting means, the second antibody will preferably havean associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease or peroxidase-conjugated anti-human IgG for aperiod of time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

The preceding format may be altered by first binding the sample to theassay plate. Then, primary antibody is incubated with the assay plate,followed by detecting of bound primary antibody using a labeled secondantibody with specificity for the primary antibody.

The steps of various other useful immunodetection methods have beendescribed in the scientific literature, such as, e.g., Nakamura et al.(1987; incorporated herein by reference). Immunoassays, in their mostsimple and direct sense, are binding assays. Certain preferredimmunoassays are the various types of radioimmunoassays (RIA) andimmunobead capture assay. Immunohistochemical detection using tissuesections also is particularly useful. However, it will be readilyappreciated that detection is not limited to such techniques, andWestern blotting, dot blotting, FACS analyses, and the like also may beused in connection with the present invention.

The antibody compositions of the present invention will find great usein immunoblot or Western blot analysis. The antibodies may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof. In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background.Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard. U.S. patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody or a biotin/avidin ligand binding arrangement, asis known in the art.

V. Methods for Screening Active Compounds

The present invention also contemplates the use of TS10q23.3 and activefragments, and nucleic acids coding therefor, in the screening ofcompounds for activity in either stimulating TS10q23.3 activity,overcoming the lack of TS10q23.3 or blocking the effect of a mutantTS10q23.3 molecule. These assays may make use of a variety of differentformats and may depend on the kind of “activity” for which the screen isbeing conducted. Contemplated functional “read-outs” include binding toa compound, inhibition of binding to a substrate, ligand, receptor orother binding partner by a compound, phosphatase activity,anti-phosphatase activity, phosphorylation of TS10q23.3,dephosphorylation of TS10q23.3, inhibition or stimulation ofcell-to-cell signaling, growth, metastasis, cell division, cellmigration, soft agar colony formation, contact inhibition, invasiveness,angiogenesis, apoptosis, tumor progression or other malignant phenotype.

The polypeptide of the invention may also be used for screeningcompounds developed as a result of combinatorial library technology.Combinatorial library technology provides an efficient way of testing apotential vast number of different substances for ability to modulateactivity of a polypeptide. Such libraries and their use are known in theart. The use of peptide libraries is preferred. See, for example, WO97/02048.

Briefly, a method of screening for a substance which modulates activityof a polypeptide may include contacting one or more test substances withthe polypeptide in a suitable reaction medium, testing the activity ofthe treated polypeptide and comparing that activity with the activity ofthe polypeptide in comparable reaction medium untreated with the testsubstance or substances. A difference in activity between the treatedand untreated polypeptides is indicative of a modulating effect of therelevant test substance or substances.

Prior to or as well as being screened for modulation of activity, testsubstances may be screened for ability to interact with the polypeptide,e.g., in a yeast two-hybrid system (e.g., Bartel et al., 1993; Fieldsand Song, 1989; Chevray and Nathans, 1992; Lee et al., 1995). Thissystem may be used as a coarse screen prior to testing a substance foractual ability to modulate activity of the polypeptide. Alternatively,the screen could be used to screen test substances for binding to anKVLQT1 or KCNE1 specific binding partner, or to find mimetics of theKVLQT1 or KCNE1 polypeptide.

Following identification of a substance which modulates or affectspolypeptide activity, the substance may be investigated further.Furthermore, it may be manufactured and/or used in preparation, i.e.,manufacture or formulation, or a composition such as a medicament,pharmaceutical composition or drug. These may be administered toindividuals.

Thus, the present invention extends in various aspects not only to asubstance identified using a nucleic acid molecule as a modulator ofpolypeptide activity, in accordance with what is disclosed herein, butalso a pharmaceutical composition, medicament, drug or other compositioncomprising such a substance, a method comprising administration of sucha composition comprising such a substance, a method comprisingadministration of such a composition to a patient, e.g., for treatment(which may include preventative treatment) of LQT, use of such asubstance in the manufacture of a composition for administration, e.g.,for treatment of LQT, and a method of making a pharmaceuticalcomposition comprising admixing such a substance with a pharmaceuticallyacceptable excipient, vehicle or carrier, and optionally otheringredients.

A. In Vitro Assays

In one embodiment, the invention is to be applied for the screening ofcompounds that bind to the TS10q23.3 molecule or fragment thereof. Thepolypeptide or fragment may be either free in solution, fixed to asupport, expressed in or on the surface of a cell. Either thepolypeptide or the compound may be labeled, thereby permittingdetermining of binding.

In another embodiment, the assay may measure the inhibition of bindingof TS10q23.3 to a natural or artificial substrate or binding partner.Competitive binding assays can be performed in which one of the agents(TS10q23.3, binding partner or compound) is labeled. Usually, thepolypeptide will be the labeled species. One may measure the amount offree label versus bound label to determine binding or inhibition ofbinding.

Another technique for high throughput screening of compounds isdescribed in WO 84/03564. Large numbers of small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with TS10q23.3 andwashed. Bound polypeptide is detected by various methods.

Purified TS10q23.3 can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to immobilize the polypeptideto a solid phase. Also, fusion proteins containing a reactive region(preferably a terminal region) may be used to link the TS10q23.3 activeregion to a solid phase.

Various cell lines containing wild-type or natural or engineeredmutations in TS10q23.3 can be used to study various functionalattributes of TS10q23.3 and how a candidate compound affects theseattributes. Methods for engineering mutations are described elsewhere inthis document, as are naturally-occurring mutations in TS10q23.3 thatlead to, contribute to and/or otherwise cause malignancy. In suchassays, the compound would be formulated appropriately, given itsbiochemical nature, and contacted with a target cell. Depending on theassay, culture may be required. The cell may then be examined by virtueof a number of different physiologic assays. Alternatively, molecularanalysis may be performed in which the function of TS10q23.3, or relatedpathways, may be explored. This may involve assays such as those forprotein expression, enzyme function, substrate utilization,phosphorylation states of various molecules including TS10q23.3, cAMPlevels, mRNA expression (including differential display of whole cell orpolyA RNA) and others.

B. In Vivo Assays

The present invention also encompasses the use of various animal models.Here, the identity seen between human and mouse TS10q23.3 provides anexcellent opportunity to examine the function of TS10q23.3 in a wholeanimal system where it is normally expressed. By developing or isolatingmutant cells lines that fail to express normal TS10q23.3, one cangenerate cancer models in mice that will be highly predictive of cancersin humans and other mammals. These models may employ the orthotopic orsystemic administration of tumor cells to mimic primary and/ormetastatic cancers. Alternatively, one may induce cancers in animals byproviding agents known to be responsible for certain events associatedwith malignant transformation and/or tumor progression. Finally,transgenic animals (discussed below) that lack a wild-type TS10q23.3 maybe utilized as models for cancer development and treatment.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route the could be utilized for clinical or non-clinicalpurposes, including but not limited to oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumoral injection.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, survival, reduction of tumor burden or mass, arrest orslowing of tumor progression, elimination of tumors, inhibition orprevention of metastasis, increased activity level, improvement inimmune effector function and improved food intake.

C. Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or compounds with which they interact(agonists, antagonists, inhibitors, binding partners, etc.). By creatingsuch analogs, it is possible to fashion drugs which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for TS10q23.3 or a fragment thereof. This could beaccomplished by x-ray crystallograph, computer modeling or by acombination of both approaches. An alternative approach, “alanine scan,”involves the random replacement of residues throughout molecule withalanine, and the resulting affect on function determined.

It also is possible to isolate a TS10q23.3 specific antibody, selectedby a functional assay, and then solve its crystal structure. Inprinciple, this approach yields a pharmacore upon which subsequent drugdesign can be based. It is possible to bypass protein crystallographaltogether by generating anti-idiotypic antibodies to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of anti-idiotype would be expected to be an analog ofthe original antigen. The anti-idiotype could then be used to identifyand isolate peptides from banks of chemically- or biologically-producedpeptides. Selected peptides would then serve as the pharmacore.Anti-idiotypes may be generated using the methods described herein forproducing antibodies, using an antibody as the antigen.

Thus, one may design drugs which have improved TS10q23.3 activity orwhich act as stimulators, inhibitors, agonists, antagonists or TS10q23.3or molecules affected by TS10q23.3 function. By virtue of theavailability of cloned TS0q23.3 sequences, sufficient amounts ofTS10q23.3 can be produced to perform crystallographic studies. Inaddition, knowledge of the polypeptide sequences permits computeremployed predictions of structure-function relationships.

A substance identified as a modulator of polypeptide function may bepeptide or non-peptide in nature. Non-peptide “small molecules” areoften preferred for many in vivo pharmaceutical uses. Accordingly, amimetic or mimic of the substance (particularly if a peptide) may bedesigned for pharmaceutical use.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesize or where it is unsuitable for a particularmethod of administration, e.g., pure peptides are unsuitable activeagents for oral compositions as they tend to be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingis generally used to avoid randomly screening large numbers of moleculesfor a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. First, the particular parts ofthe compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g., by substituting each residue in turn. Alanine scans of peptide arecommonly used to refine such peptide motifs. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modeledaccording to its physical properties, e.g., stereochemistry, bonding,size and/or charge, using data from a range of sources, e.g.,spectroscopic techniques, x-ray diffraction data and NMR. Computationalanalysis, similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modeling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this in the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted onto it can conveniently be selected so that themimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. Alternatively, where the mimetic ispeptide-based, further stability can be achieved by cyclizing thepeptide, increasing its rigidity. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimization ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

VI. Methods for Treating 10q23.3 Related Malignancies

The present invention also involves, in another embodiment, thetreatment of cancer. The types of cancer that may be treated, accordingto the present invention, is limited only by the involvement ofTS10q23.3. By involvement, it is not even a requirement that TS10q23.3be mutated or abnormal—the overexpression of this tumor suppressor mayactually overcome other lesions within the cell. Thus, it iscontemplated that a wide variety of tumors may be treated usingTS10q23.3 therapy, including cancers of the brain (glioblastoma,astrocytoma, oligodendroglioma, ependymomas), lung, liver, spleen,kidney, lymph node, pancreas, small intestine, blood cells, colon,stomach, breast, endometrium, prostate, testicle, ovary, skin, head andneck, esophagus, bone marrow, blood or other tissue.

In many contexts, it is not necessary that the tumor cell be killed orinduced to undergo normal cell death or “apoptosis.” Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis completely blocked, however, or that some tumor regression isachieved. Clinical terminology such as “remission” and “reduction oftumor” burden also are contemplated given their normal usage.

A. Genetic Based Therapies

One of the therapeutic embodiments contemplated by the present inventorsis the intervention, at the molecular level, in the events involved inthe tumorigenesis of some cancers. Specifically, the present inventorsintend to provide, to a cancer cell, an expression construct capable ofproviding TS10q23.3 to that cell. Because the sequence homology betweenthe human, mouse and dog genes, any of these nucleic acids could be usedin human therapy, as could any of the gene sequence variants discussedabove which would encode the same, or a biologically equivalentpolypeptide. The lengthy discussion of expression vectors and thegenetic elements employed therein is incorporated into this section byreference. Particularly preferred expression vectors are viral vectorssuch as adenovirus, adeno-associated virus, herpesvirus, vaccinia virusand retrovirus. Also preferred is liposomally-encapsulated expressionvector.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient.Similar figures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

Various routes are contemplated for various tumor types. The sectionbelow on routes contains an extensive list of possible routes. Forpractically any tumor, systemic delivery is contemplated. This willprove especially important for attacking microscopic or metastaticcancer. Where discrete tumor mass may be identified, a variety ofdirect, local and regional approaches may be taken. For example, thetumor may be directly injected with the expression vector. A tumor bedmay be treated prior to, during or after resection. Following resection,one generally will deliver the vector by a catheter left in placefollowing surgery. One may utilize the tumor vasculature to introducethe vector into the tumor by injecting a supporting vein or artery. Amore distal blood supply route also may be utilized.

In a different embodiment, ex vivo gene therapy is contemplated. Thisapproach is particularly suited, although not limited, to treatment ofbone marrow associated cancers. In an ex vivo embodiment, cells from thepatient are removed and maintained outside the body for at least someperiod of time. During this period, a therapy is delivered, after whichthe cells are reintroduced into the patient; hopefully, any tumor cellsin the sample have been killed.

Autologous bone marrow transplant (ABMT) is an example of ex vivo genetherapy. Basically, the notion behind ABMT is that the patient willserve as his or her own bone marrow donor. Thus, a normally lethal doseof irradiation or chemotherapeutic may be delivered to the patient tokill tumor cells, and the bone marrow repopulated with the patients owncells that have been maintained (and perhaps expanded) ex vivo. Because,bone marrow often is contaminated with tumor cells, it is desirable topurge the bone marrow of these cells. Use of gene therapy to accomplishthis goal is yet another way TS10q23.3 may be utilized according to thepresent invention.

Gene transfer systems known in the art may be useful in the practice ofthe gene therapy methods of the present invention. These include viraland nonviral transfer methods. A number of viruses have been used asgene transfer vectors or as the basis for repairing gene transfervectors, including papovaviruses (e.g., SV40, Madzak et al., 1992),adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian,1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson andAkrigg, 1992; Stratford-Perricaudet et al., 1990; Schneider et al.,1998), vaccinia virus (Moss, 1992; Moss, 1996), adeno-associated virus(Muzyczka, 1992; Ohi et al., 1990; Russell and Hirata, 1998),herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al.,1992; Fink et al., 1992; Breakefield and Geller, 1987; Freese et al.,1990; Fink et al., 1996), lentiviruses (Naldini et al., 1996), Sindbisand Semliki Forest virus (Berglund et al., 1993), and retroviruses ofavian (Bandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine(Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann andBaltimore, 1985; Miller et al., 1988), and human origin (Shimada et al.,1991; Helseth et al., 1990; Page et al., 1990; Buchschacher andPanganiban, 1992).

Nonviral gene transfer methods known in the art include chemicaltechniques such as calcium phosphate coprecipitation (Graham and van derEb, 1973; Pellicer et al., 1980); mechanical techniques, for examplemicroinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster etal., 1981; Costantini and Lacy, 1981); membrane fusion-mediated transfervia liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda etal., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1991);and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al.,1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989; Wolff etal., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al.,1990; Curiel et al., 1992; Curiel et al., 1991). Viral-mediated genetransfer can be combined with direct in vivo gene transfer usingliposome delivery, allowing one to direct the viral vectors to the tumorcells and not into the surrounding nondividing cells. Alternatively, theretroviral vector producer cell line can be injected into tumors (Culveret al., 1992). Injection of producer cells would then provide acontinuous source of vector particles. This technique has been approvedfor use in humans with inoperable brain tumors.

In an approach which combines biological and physical gene transfermethods, plasmid DNA of any size is combined with apolylysine-conjugated antibody specific to the adenovirus hexon protein,and the resulting complex is bound to an adenovirus vector. Thetrimolecular complex is then used to infect cells. The adenovirus vectorpermits efficient binding, internalization, and degradation of theendosome before the coupled DNA is damaged. For other techniques for thedelivery of adenovirus based vectors see Schneider et al. (1998) andU.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500.

Liposome/DNA complexes have been shown to be capable of mediating directin vivo gene transfer. While in standard liposome preparations the genetransfer process is nonspecific, localized in vivo uptake and expressionhave been reported in tumor deposits, for example, following direct insitu administration (Nabel, 1992).

Expression vectors in the context of gene therapy are meant to includethose constructs containing sequences sufficient to express apolynucleotide that has been cloned therein. In viral expressionvectors, the construct contains viral sequences sufficient to supportpackaging of the construct. If the polynucleotide encodes a TS10q23.3gene, expression will produce the corresponding protein. If thepolynucleotide encodes an antisense polynucleotide or a ribozyme,expression will produce the antisense polynucleotide or ribozyme. Thusin this context, expression does not require that a protein product besynthesized. In addition to the polynucleotide cloned into theexpression vector, the vector also contains a promoter functional ineukaryotic cells. The cloned polynucleotide sequence is under control ofthis promoter. Suitable eukaryotic promoters include those describedabove. The expression vector may also include sequences, such asselectable markers and other sequences described herein.

B. Immunotherapies

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

According to the present invention, it is unlikely that TS10q23.3 couldserve as a target for an immune effector given that (i) it is unlikelyto be expressed on the surface of the cell and (ii) that the presence,not absence, of TS10q23.3 is associated with the normal state. However,it is possible that particular mutant forms of TS10q23.3 may be targetedby immunotherapy, either using antibodies, antibody conjugates or immuneeffector cells.

A more likely scenario is that immunotherapy could be used as part of acombined therapy, in conjunction with TS10q23.3-targeted gene therapy.The general approach for combined therapy is discussed below. Generally,the tumor cell must bear some marker that is amenable to targeting,i.e., is not present on the majority of other cells. Many tumor markerexist and any of these may be suitable for targeting in the context ofthe present invention. Common tumor markers include carcinoembryonicantigen, prostate specific antigen, urinary tumor associated antigen,fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl LewisAntigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb Band p155.

Immunoconjugates. The invention further provides immunotoxins in whichan antibody that binds to a cancer marker, such as a mutant TS10q23.3,is linked to a cytotoxic agent. Immunotoxin technology is fairlywell-advanced and known to those of skill in the art. Immunotoxins areagents in which the antibody component is linked to another agent,particularly a cytotoxic or otherwise anticellular agent, having theability to kill or suppress the growth or cell division of cells.

As used herein, the terms “toxin” and “toxic moiety” are employed torefer to any cytotoxic or otherwise anticellular agent that has such akilling or suppressive property. Toxins are thus pharmacologic agentsthat can be conjugated to an antibody and delivered in an active form toa cell, wherein they will exert a significant deleterious effect.

The preparation of immunotoxins is, in general, well known in the art(see, e.g., U.S. Pat. No. 4,340,535, incorporated herein by reference).It also is known that while IgG based immunotoxins will typicallyexhibit better binding capability and slower blood clearance than theirFab′ counterparts, Fab′ fragment-based immunotoxins will generallyexhibit better tissue penetrating capability as compared to IgG basedimmunotoxins.

Exemplary anticellular agents include chemotherapeutic agents,radioisotopes as well as cytotoxins. Example of chemotherapeutic agentsare hormones such as steroids; antimetabolites such as cytosinearabinoside, fluorouracil, methotrexate or aminopterin; anthracycline;mitomycin C; vinca alkaloids; demecolcine; etoposide; mithramycin; oralkylating agents such as chlorambucil or melphalan.

Preferred immunotoxins often include a plant-, fungal- orbacterial-derived toxin, such as an A chain toxin, a ribosomeinactivating protein, α-sarcin, aspergillin, restirictocin, aribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention justa few examples. The use of toxin-antibody constructs is well known inthe art of immunotoxins, as is their attachment to antibodies. Ofcourse, combinations of the various toxins could also be coupled to oneantibody molecule, thereby accommodating variable or even enhancedcytotoxicity.

One type of toxin for attachment to antibodies is ricin, withdeglycosylated ricin A chain being particularly preferred. As usedherein, the term “ricin” is intended to refer to ricin prepared fromboth natural sources and by recombinant means. Various ‘recombinant’ or‘genetically engineered’ forms of the ricin molecule are known to thoseof skill in the art, all of which may be employed in accordance with thepresent invention.

Deglycosylated ricin A chain (dgA) is preferred because of its extremepotency, longer half-life, and because it is economically feasible tomanufacture it a clinical grade and scale (available commercially fromInland Laboratories, Austin, Tex.). Truncated ricin A chain, from whichthe 30 N-terminal amino acids have been removed by Nagarase (Sigma),also may be employed.

Linking or coupling one or more toxin moieties to an antibody may beachieved by a variety of mechanisms, for example, covalent binding,affinity binding, intercalation, coordinate binding and complexation.Preferred binding methods are those involving covalent binding, such asusing chemical cross-linkers, natural peptides or disulfide bonds.

The covalent binding can be achieved either by direct condensation ofexisting side chains or by the incorporation of external bridgingmolecules. Many bivalent or polyvalent agents are useful in couplingprotein molecules to other proteins, peptides or amine functions.Examples of coupling agents are carbodiimides, diisocyanates,glutaraldehyde, diazobenzenes, and hexamethylene diamines. This list isnot intended to be exhaustive of the various coupling agents known inthe art but, rather, is exemplary of the more common coupling agentsthat may be used.

In preferred embodiments, it is contemplated that one may wish to firstderivatize the antibody, and then attach the toxin component to thederivatized product. As used herein, the term “derivatize” is used todescribe the chemical modification of the antibody substrate with asuitable cross-linking agent. Examples of cross-linking agents for usein this manner include the disulfide-bond containing linkers SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate) and SMPT(4-succinimidyl-oxycarbonyl-α-methyl-α(2-pyridyldithio)toluene).

Biologically releasable bonds are particularly important to therealization of a clinically active immunotoxin in that the toxin moietymust be capable of being released from the antibody once it has enteredthe target cell. Numerous types of linking constructs are known,including simply direct disulfide bond formation between sulfhydrylgroups contained on amino acids such as cysteine, or otherwiseintroduced into respective protein structures, and disulfide linkagesusing available or designed linker moieties.

Numerous types of disulfide-bond containing linkers are known which cansuccessfully be employed to conjugate toxin moieties to antibodies,however, certain linkers are generally preferred, such as, for example,sterically hindered disulfide bond linkers are preferred due to theirgreater stability in vivo, thus preventing release of the toxin moietyprior to binding at the site of action. A particularly preferredcross-linking reagent is SMPT, although other linkers such as SATA, SPDPand 2-iminothiolane also may be employed.

Once conjugated, it will be important to purify the conjugate so as toremove contaminants such as unconjugated A chain or antibody. It isimportant to remove unconjugated A chain because of the possibility ofincreased toxicity. Moreover, it is important to remove unconjugatedantibody to avoid the possibility of competition for the antigen betweenconjugated and unconjugated species. In any event, a number ofpurification techniques have been found to provide conjugates to asufficient degree of purity to render them clinically useful.

In general, the most preferred technique will incorporate the use ofBlue-Sepharose with a gel filtration or gel permeation step.Blue-Sepharose is a column matrix composed of Cibacron Blue 3GA andagarose, which has been found to be useful in the purification ofimmunoconjugates. The use of Blue-Sepharose combines the properties ofion exchange with A chain binding to provide good separation ofconjugated from unconjugated binding. The Blue-Sepharose allows theelimination of the free (non conjugated) antibody from the conjugatepreparation. To eliminate the free (unconjugated) toxin (e.g., dgA) amolecular exclusion chromatography step may be used using eitherconventional gel filtration procedure or high performance liquidchromatography.

After a sufficiently purified conjugate has been prepared, one willgenerally desire to prepare it into a pharmaceutical composition thatmay be administered parenterally. This is done by using for the lastpurification step a medium with a suitable pharmaceutical composition.Such formulations will typically include pharmaceutical buffers, alongwith excipients, stabilizing agents and such like. The pharmaceuticallyacceptable compositions will be sterile, non-immunogenic andnon-pyrogenic. Details of their preparation are well known in the artand are further described herein. It will be appreciated that endotoxincontamination should be kept minimally at a safe level, for example,less that 0.5 ng/mg protein.

Suitable pharmaceutical compositions in accordance with the inventionwill generally comprise from about 10 to about 100 mg of the desiredconjugate admixed with an acceptable pharmaceutical diluent orexcipient, such as a sterile aqueous solution, to give a finalconcentration of about 0.25 to about 2.5 mg/ml with respect to theconjugate.

As mentioned above, the antibodies of the invention may be linked to oneor more chemotherapeutic agents, such as anti-tumor drugs, cytokines,antimetabolites, alkylating agents, hormones, nucleic acids and thelike, which may thus be targeted to a TS10q23.3 expressing cancer cellusing the antibody conjugate. The advantages of antibody-conjugatedagents over their non-antibody conjugated counterparts is the addedselectivity afforded by the antibody.

In analyzing the variety of chemotherapeutic and pharmacologic agentsavailable for conjugating to an antibody, one may wish to particularlyconsider those that have been previously shown to be successfullyconjugated to antibodies and to function pharmacologically. Exemplaryantineoplastic agents that have been used include doxorubicin,daunomycin, methotrexate, vinblastine. Moreover, the attachment of otheragents such as neocarzinostatin, macromycin, trenimon and α-amanitin hasalso been described. The lists of suitable agents presented herein are,of course, merely exemplary in that the technology for attachingpharmaceutical agents to antibodies for specific delivery to tissues iswell established.

Thus, it is generally believed to be possible to conjugate to antibodiesany pharmacologic agent that has a primary or secondary amine group,hydrazide or hydrazine group, carboxyl alcohol, phosphate, or alkylatinggroup available for binding or cross-linking to the amino acids orcarbohydrate groups of the antibody. In the case of protein structures,this is most readily achieved by means of a cross linking agent, asdescribed above for the immunotoxins. Attachment also may be achieved bymeans of an acid labile acyl hydrazone or cis aconityl linkage betweenthe drug and the antibody, or by using a peptide spacer such asL-Leu-L-Ala-L-Leu-L-Ala, between the γ-carboxyl group of the drug and anamino acid of the antibody.

C. Protein Therapy

Another therapy approach is the provision, to a subject, of TS10q23.3polypeptide, active fragments, synthetic peptides, mimetics or otheranalogs thereof. The protein may be produced by recombinant expressionmeans or, if small enough, generated by an automated peptidesynthesizer. Formulations would be selected based on the route ofadministration and purpose including, but not limited to, liposomalformulations and classic pharmaceutical preparations.

D. Combined Therapy with Immunotherapy, Traditional Chemo- orRadiotherapy

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy. For example, theherpes simplex-thymidine kinase (HS-tk) gene, when delivered to braintumors by a retroviral vector system, successfully inducedsusceptibility to the antiviral agent ganciclovir (Culver et al., 1992).In the context of the present invention, it is contemplated thatTS10q23.3 replacement therapy could be used similarly in conjunctionwith chemo- or radiotherapeutic intervention. It also may proveeffective to combine TS10q23.3 gene therapy with immunotherapy, asdescribed above.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentinvention, one would generally contact a “target” cell with a TS10q23.3expression construct and at least one other agent. These compositionswould be provided in a combined amount effective to kill or inhibitproliferation of the cell. This process may involve contacting the cellswith the expression construct and the agent(s) or factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent.

Alternatively, the gene therapy treatment may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and expression construct are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and expression construct would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hours of each other and, more preferably, within about 6-12hours of each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

It also is conceivable that more than one administration of eitherTS10q23.3 or the other agent will be desired. Various combinations maybe employed, where TS10q23.3 is “A” and the other agent is “B”, asexemplified below:

-   -   A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B    -   A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A    -   A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are contemplated. Again, to achieve cell killing,both agents are delivered to a cell in a combined amount effective tokill the cell.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic agents,” function toinduce DNA damage, all of which are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated to be of use, include, e.g., adriamycin, 5-fluorouracil(5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. The invention alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide. In certainembodiments, the use of cisplatin in combination with a TS10q23.3expression construct is particularly preferred as this compound.

In treating cancer according to the invention, one would contact thetumor cells with an agent in addition to the expression construct. Thismay be achieved by irradiating the localized tumor site with radiationsuch as X-rays, UV-light, γ-rays or even microwaves. Alternatively, thetumor cells may be contacted with the agent by administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound such as, adriamycin, 5-fluorouracil,etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably,cisplatin. The agent may be prepared and used as a combined therapeuticcomposition, or kit, by combining it with a TS10q23.3 expressionconstruct, as described above.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with TS10q23.3. Agents such as cisplatin, andother DNA alkylating agents may be used. Cisplatin has been widely usedto treat cancer, with efficacious doses used in clinical applications of20 mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventors propose that the regional delivery of TS10q23.3 expressionconstructs to patients with 10q23.3-linked cancers will be a veryefficient method for delivering a therapeutically effective gene tocounteract the clinical disease. Similarly, the chemo- or radiotherapymay be directed to a particular, affected region of the subjects body.Alternatively, systemic delivery of expression construct and/or theagent may be appropriate in certain circumstances, for example, whereextensive metastasis has occurred.

In addition to combining TS10q23.3-targeted therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of TS10q23.3 andp53 or p16 mutations at the same time may produce an improvedanti-cancer treatment. Any other tumor-related gene conceivably can betargeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2,BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc,neu, raf, erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating a TS10q23.3. In this regard,reference to chemotherapeutics and non-TS10q23.3 gene therapy incombination should also be read as a contemplation that these approachesmay be employed separately.

E. Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—expression vectors, virus stocks,proteins, antibodies and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well know inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

VII. Transgenic Animals/Knockout Animals

In one embodiment of the invention, transgenic animals are producedwhich contain a functional transgene encoding a functional TS10q23.3polypeptide or variants thereof. Transgenic animals expressing TS10q23.3transgenes, recombinant cell lines derived from such animals andtransgenic embryos may be useful in methods for screening for andidentifying agents that induce or repress function of TS10q23.3.Transgenic animals of the present invention also can be used as modelsfor studying indications such as cancers.

In one embodiment of the invention, a TS10q23.3 transgene is introducedinto a non-human host to produce a transgenic animal expressing a humanor murine TS10q23.3 gene. The transgenic animal is produced by theintegration of the transgene into the genome in a manner that permitsthe expression of the transgene. Methods for producing transgenicanimals are generally described by Wagner and Hoppe (U.S. Pat. No.4,873,191; which is incorporated herein by reference), Brinster et al.1985; which is incorporated herein by reference in its entirety) and in“Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition (eds.,Hogan, Beddington, Costantimi and Long, Cold Spring Harbor LaboratoryPress, 1994; which is incorporated herein by reference in its entirety).

It may be desirable to replace the endogenous TS10q23.3 by homologousrecombination between the transgene and the endogenous gene; or theendogenous gene may be eliminated by deletion as in the preparation of“knock-out” animals. Typically, a TS10q23.3 gene flanked by genomicsequences is transferred by microinjection into a fertilized egg. Themicroinjected eggs are implanted into a host female, and the progeny arescreened for the expression of the transgene. Transgenic animals may beproduced from the fertilized eggs from a number of animals including,but not limited to reptiles, amphibians, birds, mammals, and fish.Within a particularly preferred embodiment, transgenic mice aregenerated which overexpress TS10q23.3 or express a mutant form of thepolypeptide. Alternatively, the absence of a TS10q23.3 in “knock-out”mice permits the study of the effects that loss of TS10q23.3 protein hason a cell in vivo. Knock-out mice also provide a model for thedevelopment of TS10q23.3-related cancers.

Methods for producing knockout animals are generally described byShastry (1995, 1998) and Osterrieder and Wolf (1998). The production ofconditional knockout animals, in which the gene is active until knockedout at the desired time is generally described by Feil et al. (1996),Gagneten et al. (1997) and Lobe and Nagy (1998). Each of thesereferences is incorporated herein by reference.

As noted above, transgenic animals and cell lines derived from suchanimals may find use in certain testing experiments. In this regard,transgenic animals and cell lines capable of expressing wild-type ormutant TS10q23.3 may be exposed to test substances. These testsubstances can be screened for the ability to enhance wild-typeTS10q23.3 expression and or function or impair the expression orfunction of mutant TS10q23.3.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skilled the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Homozygous Deletions in Glioma Cell Lines

The inventors have examined DNA from a series of 21 glioma cell linesand primary cultures, along with normal cells, to identify homozygousdeletions of genomic material on chromosome 10. Markers were chosen fortheir approximate location at or near previously implicated regions(FIG. 1). The cells analyzed were generated in the Department ofNeuro-Oncology UTMDACC (LG11, EFC-2, PL-1, PC-1, JW, FG-2, FG-0, NG-1,PH-2, KE, PC-3, and D77), were commercially available (U138, A172, U373,U87, U251, U118, and T98G), or obtained from collaborators (13 wk astro,D54-MG). Markers were obtained from Research Genetics, Huntsville, Ala.,or synthesized from reported sequence. Once cell line, EFC-2, revealed alarge homozygous deletion associated with four markers surroundingD10S215 (FIG. 2). This deletion was also observed by FISH using YAC746h6, which maps to the region. Three other cell lines (D-54, A172, andLG11) also demonstrated homozygous deletions at AFM086, thereby stronglyimplicating the region to contain a putative tumor suppressor gene (FIG.2). Deletions in PCR™ reactions were performed in the presence of twoprimer pairs (multiplexed) to assure appropriate amplificationconditions. All deletions were confirmed by (at least) triplicatereactions. This same region has also been implicated in prostatecarcinoma (Gray et al., 1995). Homozygous deletions in cell lines alsohave been used to define a tumor suppressor gene locus at 3p21.3 insmall cell lung carcinoma (Daly et al., 1993; Kok et al., 1994; Wei etal., 1996).

Example 2 Retention of 10q Loci in Suppressed Hybrid Cells

The inventors' second strategy was to examine the regions of chromosome10 that were retained in suppressed hybrid clones, but absent in therevertant clones. This analysis extended the inventors' previous study,showing the presence of two tumor suppressor loci on chromosome 10 andanalyzing the regions that were retained. Hybrids retaining all orportions of 10q failed to grow in soft agarose and in nude mice (“fully”suppressed clones), while hybrid cells that lost the majority of theinserted chromosome 10q grew in soft agarose, but were nontumorigenic(“partially” suppressed clones; Steck et al., 1995; FIG. 3, right side).Original clones U251N10.6, N10.7, and N10.8 previously were shown toretain only fragments of 10q (Pershouse et al., 1993; Steck et al.,1995). Using additional informative microsatellite markers, threeretained regions were identified in all three suppressed clones; a 22 cMregion from D10S219 to D10S110, a 14 cM region from D10S192 to D10S187,and a 18 cM region from D10S169 through D10S1134 (FIG. 3).

To bypass this limitation, the originally transferred neomycinresistance-tagged chromosome 10 from hybrid U251.N10.7 was “rescued” bymicrocell-mediated chromosome transfer into mouse A9 cells. This allowsall human microsatellite markers to be informative for the presence ofchromosome 10. The basis for this analysis is that all “fully”suppressed subclones should retain a common region and this region isdeleted in the “partially” suppressed subclones. An additional impetuswas that N10.7 displayed considerable heterogeneity in the size ofchromosome 10 retained, as determined by FISH using chromosome 10specific probes. Also, hybrid cells used for this rescue were firstassayed for soft agarose growth and showed no colony formation. Themouse hybrids containing the transferred human chromosome 10 allcontained the short arm of chromosome 10. The same region was retainedin the “partially” suppressed clones (N10.5a-j) that grew in softagarose (Steck et al., 1995), thus excluding this region (10pter-10q11)as containing the 10q tumor suppressor gene. Examination of the retainedregions of 10q illustrated considerable heterogeneity (FIG. 3). Themajority of clones showed either partial or extensive deletions of10q23-26. Only two regions were retained in all the subclones examined.The most centromeric region retained involved the markers D10S210 andD10S219. However, these markers were absent in the original N10.6 and/orN10.8 clones, excluding this region (FIG. 3). The other region wascentromeric of D4S536 but telomeric of D10S215 (˜4 cM). The markersAFM086 and D10S536 were retained in all clones examined (boxed region inFIG. 3). These markers were absent in the partially suppressed clones(N10.5a-j). These results demonstrate that a common region, surroundingAFM086, is retained in all hybrid cells that are phenotypicallysuppressed. This same region is deleted in several glioma cell lines.

This analysis has several limitations. First, the rescued clones cannotbe analyzed for biological activity, therefore any changes in chromosome10 which may have occurred during or after transfer could not bedetected. To partially address this concern, the inventors' analysis wasperformed as soon as the clones were able to be harvested. Furthermore,retention of this portion of the chromosome may only “correct” an invitro artifactual deletion. Consequently, allelic deletion studies wereperformed to determine if this region was involved in gliomas. Also, analternative region was suggested by this analysis at D10S1158, where allthe clones but one (C7) retained this region. However, the retainedregion at AFM086 also exhibited homozygous deletions, thereby beingimplicated by two alternative methods as compared to D10S1158. It isalso interesting to note that the tumor suppressor gene region appearsto be preferentially retained, while the remainder of 10q is fragmented.

Example 3 Allelic Deletion Analysis of 10q

An allelic deletion study was performed on DNA from a series of 53glioma specimens and corresponding patient lymphocytes usingmicrosatellite markers specific for chromosome 10. This study wasundertaken to determine if our critical region also was involved inglioma specimens. Extensive deletions were observed in the majority ofspecimens derived from GBM, with 30 of 38 GBMs exhibiting deletion ofmost or all of chromosome 10 markers. Less extensive deletions wereobserved in the majority if specimens derived from anaplasticastrocytomas, while infrequent deletions were observed in astrocytomasand most oligodendrogliomas (FIG. 4 and data not shown). The majority ofmarkers used in this analysis mapped to 10q23-26 (Gyapay et al., 1994).Similar to other studies, a common region of deletion could not beconvincingly demonstrated, due to the large deletions in most GBMsamples (Fults et al., 1993; Rasheed et al., 1995).

However, for the GBM specimens examined, all but one tumor sample (#9;FIG. 4) revealed deletions involving the region from D10S579 to D10S541.Furthermore, only one AA showed a deletion at the inventors' criticalregion, and no astrocytomas. Two oligodendrogliomas exhibited deletionswithin the critical region, but both were diagnosed as malignant. Thisstudy presents several possibilities. First, the deletions involving theinventors' critical region occur predominantly in GBMs and not in lowergrade tumors. This would imply that loss of the tumor suppressor gene onchromosome 10q in the inventors' critical region would represent agenetic alteration associated with progression to GBM. In support ofthis hypothesis, even though deletions occur on 10q in lower gradetumors, no common region of deletion on 10q was identified for thesespecimens. This observations would, again, support the inventors'previous suggestion that deletion of the 10q tumor suppressor gene ispredominantly associated with GBMs and not all deletions on 10q affectthe tumor suppressor gene. The region D10S216 to D10S587, showedextensive deletions, but several GBMs exhibited retention ofheterozygosity at this region (tumors #2, #9, #13, #26; FIG. 4). Also,if low grade tumors are excluded from their study, the inventors' regionis implicated in all GBMs. This combination of independent approachesstrongly suggests a 10q tumor suppressor gene maps to the region D10S215to D10S541, specifically at AFM086.

Example 4 Mapping of Candidate Tumor Suppressor Gene Region

The critical region the inventors have identified is centered at AFM086and is bordered by D10S215 and S10S541 (FIGS. 2 and 8). This region isrelatively small, being contained within several individual YACs (787d7;746h8; 934d3). FISH painting with YAC 746h8 on EFC-2 metaphase spreadsshows that the homozygous deletion is contained within the YAC as theYAC was partially observed and adjacent YACs on both sides were present.Bacterial artificial chromosomes (BACs) or PACs for all markers in theregion have been isolated (FIG. 8). The BAC contig of the region wasconstructed from end sequences of BACs mapping to the region. Severalnotable features have been identified. First, two overlapping BACs wereidentified (46b12 and 2f20) and verify the genomic integrity of 106d16.Second, a Not I site was identified at one end of the BACs. The presenceof the Not I site and coincident restriction digestion with SacII, EagI,and BssHII suggest the presence of a CpG island within 106d16.

The EcoRI fragments from BAC 106d16 were used to examine the extent ofthe homozygous deletions, by Southern blotting, in the glioma cells thatwere previously shown to have homozygously deleted AFMO86 (FIGS. 2 and5). The right side (EcoRI fragment 14) contains the probable CpG islandand is present in three of the four cell lines. A NotI/EcoRI (#3)fragment was used as a probe on a Southern blot containing several BACsand the glioma cell line (FIG. 2). Deletions to the telomeric side(right side) have not been detected using probes from 46b12, except forEFC-2 cells. However, additional homozygous deletions have been observedin the cells within the region defined by 106d16 (˜65 kb). A homozygousdeletion for band 3 is observed for LG11 and EFC-2 cells, but not theadditional glioma cells or normal controls. 106d16 (band 12) has beenobserved to be present in all cells (EFC-2 exhibits an altered migratingband), suggesting the homozygous deletion is contained entirely within106d16.

Example 5 Identification of Expressed Genes within the Critical Region

EcoRI fragments from BAC 106d 16 were generated and size separated byagarose gel electrophoresis. Individual bands or pools of similar sizedbands were ligated into pSPL3 (GIBCO, Gaithersburg, Md.). Putative exonswere identified as described by the manufacturer. Two exons wereproperly spliced into the trapping vector. The exons were derived fromband pool 2, 3, 4, 5 and band 7. The sequence of the trapped exons wasdetermined and defined by the known trapping vector sequence. UsingBLAST searches of expressed sequence tag (dbEST) database, fivepotential expressed sequence tags (ESTs) were identified. Two ESTs(gb/H92038, AA009519) were observed to contain either one or both of theexons (albeit one EST was in the wrong orientation).

Sequencing primers were generated from the ESTs and used to defineputative exon-intron boundaries using BAC46b12 as a template. Nine exonswere identified. Sequence differences between the ESTs and the genomictemplate were corrected. All the exons were contained within BAC 46b12.Primers were generated from the intron sequences adjacent to the exonsto form amplicon units for each exon. Two of the exons were correspondedto the trapped exons from the BAC 106d16 EcoRI sequences. The sequenceof the gene is shown in FIG. 6. The predicted amino acid reading wasdefined by the presence of an ATG start site, TGA and TAA stop codons inframe, the presence of multiple stop codons in all three reading frameselsewhere in the sequence, nine splicing sites, and the presence ofKozak signals near the initiation site. The 403 amino acid sequence isshown in FIG. 7 and FIG. 9. The predicted molecular weight is 47,122with a pI of 5.86.

A possible functional role for the protein product is suggested by itssequence homology to several protein motifs. A critical motif fromresidues 88 to 98 [IHCKAGKGRTG](SEQ ID NO:28) has an exact match for theconserved catalytic domain of a protein tyrosine phosphatase[(I/V)HCxAGxxR(S/T)G] (SEQ ID NO:29; Denu et al., 1996). Several othermotifs were identified that would agree with the phosphatase functionfor the tumor suppressor gene.

Amplicons (PCR™ products generated from various regions of the gene)were generated from random primed cDNA. The amplicons sequencecorresponded to the DNA sequence. Non-overlapping amplicons were used toprobe Northern blots of normal tissue derived from various organs(Clontech, Palo Alto, Calif.; multitissue blots). All ampliconsidentified a major band at 5.5 to 6 kb on the Northern blots and severalminor bands. The message was expressed in all tissues examined (heart,brain, placenta, lung, liver skeletal muscle, kidney, pancreas, spleen,thymus, prostate, testes, ovary, small intestine, colon and peripheralblood lymphocytes).

Example 6 Mutational Analysis

The mutational analyses have initially proceeded on two fronts. First,the glioma cell lines initially shown to have homozygous deletions wereanalyzed for the presence of the candidate gene. As shown in FIG. 8, allof the cell lines that exhibited deletion of AFM086 had homozygousdeletions of multiple exons of the candidate gene. Furthermore, thedeletions occurred in the middle of the gene, thus defining the deletionboundaries (similar deletions in all cell lines) between exons 2 and 7.Deletions that affect the middle of the gene further indicate that theidentified gene represents the gene targeted for mutation.

Preliminary analysis for sequence mutations was also performed on aseries of glioma cell lines. Mutations and/or deletions were observed inall but three glioma cell lines examined (Table 5). Reference to basenumber in the table references the exon, not the entire sequences, i.e.,the 98th base of exon 7 for U251. TABLE 5 IDENTIFIED MUTATIONS INCANDIDATE GENE Cells Cell Type Mutation Predicted Effect 1 U87 gliomasplice junction splicing variant exon 3: G + 1 > T 2 U138 gliomasplicing site splicing variant exon 8; G + 1 > T 3 U251 glioma 2 bpaddition exon 7; 98 ins TT 4 U373 glioma frame shift exon 7 5 EFC-2glioma -all exons no product 6 D54 glioma -exons 3-9 no product 7 A172glioma -exons 3-9 no product 8 LG11 glioma -exons 2-9 no product 9 T98Gglioma missense exon 2; T46→G leu > arg 10 KE glioma missense exon 2;G28→A gly > glu 11 F60 glioma terminal mutation exon 8; terminal stopC202→T 12 D77 glioma no mutation (heterogeneous for 10q 13 PC-3 lowgrade no mutation 14 PH-2 low grade no mutation 15 nLnCap prostatedeletion exon 1, silent 16-17 del AA; mutation exon 2, C53→T

Also, deletions of exons were found in LnCap, a prostate cell line. Theglioma cells that failed to show a mutation/deletion were derived fromlow grade tumors (PC-3 and PH-2) where no allelic deletion of chromosome10 is expected and has been observed for these cells. The other cells(D77) were a primary cell culture, and chromosome 10 was shown to beheterozygous from a 1 bp polymorphism within the gene. A breast cancercell line also showed a mutation. This initial analysis supports theinventors' conclusion that loss of a 10q tumor suppressor generepresents a critical molecular marker for glioblastoma and diseaseprogression.

Example 7 Analysis of TS10Q23.3 Mutations in Cancer Specimens and TumorCell Lines

In a more extensive study, the inventors report the incidence ofTS10q23.3 mutations in 342 primary tumor specimens and 164 tumor celllines (TCLs), which exhibit apparent LOH across the TS10q23.3 locus,from various cancer types. Out of 75 TCLs that displayed apparent lossof heterozygosity (LOH) across the TS10q23.3 locus, the inventors foundten homozygous deletions that removed coding portions of TS10q23.3,along with one frameshift, one nonsense and seven missense variants. Incontrast, out of 84 primary tumors prescreened for LOH, the inventorsonly detected a frameshift lesion, a nonsense mutation, a splicingvariant and a missense variant. Of interest, the expression of TS10q23.3message was shown to be significantly reduced in high gradeglioblastomas compared to normal brain tissues.

Methods

LOH Analysis: Total genomic DNA was purified from frozen specimens ordeparaffinized sections. Total genomic DNA was purified from cancer celllines using the Easy-DNA kit (Invitrogen, San Diego, Calif.). LOHanalysis was performed as previously described (Teng et al., 1996; Stecket al., 1995). The polymorphic short tandem repeat markers used in thisstudy were: D10S1687 (heterozygosity index, H.I.=0.81; Ldb (Collins etal., 1996) radiation map location from p-telomere, R.L.=85 Mb), D10S579(H.I.=0.59; R.L.=86.4 Mb), D10S541 (H.I.=0.78; R.L.=86.5 Mb), AFM280WE1(H.I.=not determined; R.L.=87 Mb), AFMA114XB1 (H.I.=0.70; R.L.=91.9 Mb)and D10S1753 (H.I.=0.74; R.L.=92.48 Mb). TS10q23.3 as defined byAFM086WE1 is at 86.5 Mb. LOH was assessed in primary tumor specimens, inthe majority of cases, by quantitatively comparing STR marker ampliconsgenerated from tumor and normal DNAs of each individual tested. In thecase of TCLs and some primary tumors, LOH was assessed on the basis ofcombined apparent hemizygosity of AFMA114XB1, D10S541 and D10S1753; thelikelihood that all three of these STR markers are homozygous in a givensample is less than 0.017.

Homozygous Deletion Screen: Using the cell line genomic DNAs astemplates, nested PCR™ amplifications were performed with either TaqPlus(Strategene, La Jolla, Calif.) or AmpliTaq Gold (Perkin Elmer, FosterCity, Calif.). The primers used for generating TS10q23.3 and MMK4amplicons, and the PCR™ conditions used, are as described below. Twentyμl of the secondary reactions were fractionated on 2-3% Nu Sieve (FMCBioproducts) agarose gels and subsequently visualized.

Mutation screen: The inventors performed nested PCR™ amplifications ongenomic DNAs of tumor specimens or TCLs, and screened the resultingamplicons for sequence variants according to the procedures of Steck etal., (1997) with several modifications. First, exon 6 was screened witha single secondary amplicon amplified using the exon 6 FB-RR primerpair. Second, after a primary amplification of exon 8 using FA-RPprimers, the exon was screened as two secondary amplicons using thefollowing FB-RQ and FC-RR primers: CA6.ex8.FB (SEQ ID NO:33)GTTTTCCCAGTCACGACGAGGTGACAGATTTTCTTTTTTA CA6.ex8.RQ (SEQ ID NO:34)AGGAAACAGCTATGACCATTCGGTTGGCTTTGTCTTTA CA6.ex8.FC (SEQ ID NO:35)GTTTTCCCAGTCACGACGCATTTGCAGTATAGAGCGT CA6.ex8.RR (SEQ ID NO:36)AGGAAACAGCTATGACCATAGCTGTACTCCTAGAATTA

Third, since mononucleotide runs in certain introns caused poordye-primer sequencing, the inventors obtained dye-terminator sequencedata on secondary amplicons exon 8 FB-RQ and exon 9 FB-RR using thenested primers 5′-TTTTTTTTTAGGACAAAATGTTTC-3′ (SEQ ID NO:37) and5′-AATTCAGACTTTTGTAATTTGTG-3′ (SEQ ID NO:38), respectively. Theinventors obtained greater than 90% coverage of the TS10q23.3 codingsequence for all samples screened; all mutations were confirmed bysequencing a newly amplified product.

RT-PCR™ Expression: Messenger RNA was isolated from frozen sections of10 normal tissue and 10 high normal tissue and 10 high grade gliobastomaspecimens. Frozen sections (5 μm, 20 each) were cut and used to isolatemRNA (Micro-Fast Track; Invitrogen, San Diego, Calif.). Adjacentsections were histologically examined and the sections were shown tocontain predominantly normal or tumor cells. Normal sections wereobtained from regions that free from tumor during the normal course oftherapeutic craniotomies. Complementary DNA was made using SuperscriptII and primers to amplify TS10q23.3 corresponding to −28 to 347 or 345to 1232 of the coding region. The primers used were: M5′F:TCCTTTTTCTTCAGCCACAG (SEQ ID NO:39) M5′ R: ATTGCTGCAACATGATTGTC; (SEQ IDNO:40) M3′F: TGACAATCATGTTGCAGCA; (SEQ ID NO:41) F3′R:TTTATTTTCATGGTGTTTTATCC. (SEQ ID NO:42)

The PCR™ conditions were similar to those previously described exceptthe annealing step was performed at 53° C.

Characterization of a TS10q23.3 Pseudogene: DNA fragments were amplifiedfrom a human fetal brain cDNA library using Pfu polymerase and a nestedPCR™ strategy. The initial χ μl reaction contained 100 ng of cDNA. Theprimer pair used in the first round of amplification wereCTTCAGCCACAGGCTCCCAGAC (SEQ ID NO:43) and GGTGTTTTATCCCTCTTG (SEQ IDNO:44), after which the reaction was diluted 20-fold and reamplifiedwith CGGGATCCATGACAGCCATCATCAAAGAGATC (SEQ ID NO:45) andCGGAATTCTCAGACTTTTGTAATTG (SEQ ID NO:46) primers. The PCR™ conditionsused were an initial denaturation step at 94° C. for 5 min followed by30 cycles of 94° C. for 45 s, 55° C. for 30 s, and 72° C. for 1 min. Todetermine the chromosomal location of this pseudogene, the inventorsperformed radiation hybrid mapping using the Genebridge 4 panel (GenomeSystems) and the following primer pair designed to generate a specific303 bp product from the pseudogene but not TS10q23.3:ATCCTCAGTTTGTGGTCTGC (SEQ ID NO:47) and GAGCGTGCAGATAATGACAA (SEQ IDNO:48). Using this STS, the inventors determined that the pseudogene waslocated at about 160 cR on chromosome 9. Additionally, the inventorshave isolated two bacterial artificial clones (BACs), 145c22 and 188122,that carry this pseudogene and have confirmed its genomic DNA sequence.Comparison of TS10q23.3 coding sequence to that of the pseudogenerevealed the following base differences: T2G, C89T, T202C, T242C, G248A,A258G, G397A, A405T, G407A, T531C, T544G, C556G, A672G, C700T, A705G,C720T C900T and A942G. The nucleotide sequence for the human TS10q23.3pseudogene is set forth in SEQ ID NO:64.

Since TS10q23.3 appears to encode a tumor suppressor gene, theinventors' initial step toward identifying new mutations in this genewas to prescreen primary tumors and TCLs for LOH within this region of10q23. Altogether 342 primary tumor specimens and 164 TCLs were examinedfor LOH using polymorphic short tandem repeat markers on chromosome 10located near the TS10q23.3 locus (Table 6). In this panel of samples,the inventors observed LOH in primary tumor specimens at frequenciesranging from 20% in colon specimens to 75% in glioblastoma multiforms(GBMs), with an overall LOH frequency of ˜49%. For TCLs with samplesizes greater than nine, the incidence of LOH varied from 28% (colon) to82% (GBMs), with an overall frequency of ˜46%. TABLE 6 LOH ANALYSES OFTUMOR SPECIMENS AND TUMOR CELL LINES Tumor Specimens Tumor Cell LinesTumor Type LOH/screened¹ Sequenced^(2,3) LOH/screened¹ Sequenced^(2,4)Brain (Gliomas)  40/53⁵ (75%)  26⁵  9/11⁵ (82%)  7⁵ Pediatric brain  5/7 7 (2) — — Bladder — — ¾  2 Breast  32/67⁵ (48%)  31⁵ 14/22 (64%) 13Cecum — —  1/6  1 Colon  3/15⁶ (20%)  1  7/25 (28%)  7 Duodenum — —  1/1 1 Endometrial  6/13 (46%)  0 — — Head and neck  9/14 (64%)  9 — —Kidney  8/20³ (40%)  8³ — — Leukemia — — 11/23 (48%) 11 Lung  10/27(37%)  7  7/17 (41%)  6 Lymphoma — —  2/3  2 Melanoma  10/21 (48%)  15 7/14 (50%)  3 Neuroblastoma — —  0/3 — Ovarian  10/19 (52%)  9  3/8  3Pancreatic  7/19 (37%)  0  5/12 (42%)  5 Prostate  10/24 (42%)  8 (2) 1/1⁷ — Retinoblastoma — —  0/2 — Sarcomas  4/16 (25%)  6 (2) — —Submaxillary gland — —  1/1  1 Testis — —  3/5  3 Thyroid  6/17 (35%)  2 0/2 — Uterine — —  0/4 — Metastatic⁸  6/10 (60%)  8 (2) — — Total166/342⁵ (49%) 137 (8)^(5,9) 75/164 (46%) 65¹LOH percentage was only calculated for sample sizes greater than nine.²Samples that amplified and sequenced successfully (>90% coding sequencescreened).³The number of non-LOH samples that were sequenced are shown inparentheses. Certain primary tumor DNAs, particularly pancreatic andendometrial carcinomas, were isolated from microdissectedparaffin-embedded sections and failed to sequence at >90% coverage dueto poor template quality.⁴All TCLs screened displayed apparent LOH. TCLs with homozygousdeletions in the coding portion of TS10q23.3 were not screened bysequencing.⁵These totals include samples that were previously reported by Steck etal. (1997).⁶Five of these colon samples consisted of cancers that had metastasizedto the liver, although the liver metastases exhibited no LOH.⁷The prostate line, NCIH660 (TCL10F4), was characterized by Li et al.(1997) and shown to be homozygously deleted from exons 2-9 of TS10q23.3.⁸These metastatic tumor specimens originated from adenocarcinomas, asarcoma, a renal cell carcinoma and a melanoma. The metastatic lesionswere to the lung, except the melanoma which was to the groin.⁹Of these 137 specimens analyzed by sequencing, 45 had been reported(Steck et al., 1997), 8 were non-LOH and 84 displayed LOH.

To search for coding variants of TS10q23.3 in primary tumors, theinventors sequenced amplicons consisting of the exons and flankingsplice junctions of this gene amplified from tumor DNAs that displayedLOH. A caveat of this approach is that it fails to identify regulatorymutations that affect the expression levels of this gene. In addition,this screen excludes the possibility of finding mutant homozygotes andcompound heterozygotes but the incidence of these kinds of mutants ispresumably low. Previously, the inventors reported that the incidence ofTS10q23.3 coding variants in glioblastomas, breast and kidney carcinomaswere 6/26 (23%), 2/14 (14%) and 1/4, respectively (Steck et al., 1997).In this study, out of 84 primary tumors exhibiting LOH surrounding theTS10q23.3 locus, the inventors detected a frameshift mutation (breastcarcinoma), a nonsense mutation (pediatric GBM), a splicing variant(pediatric GBM) and a missense variant (melanoma; Table 7). TABLE 7TS10q23.3 Variants Identified in Primary Tumors and Tumor Cell LinesPredicted Sample Type Mutation Exon/intron Codon Effect PGT-2 Pediatricglioma¹ G > T at −1 intron 2 — splicing variant MT-1 Melanoma¹CC112-113TT exon 2 38 Pro > Phe TCL10B1 Breast T323G exon 5 108 Leu >Arg TCL10H2 Leukemia T331C exon 5 111 Trp > Arg TCL11E12 GlioblastomaT335G exon 5 112 Leu > Arg PGT-5 Pediatric glioma¹ C388T exon 5 130Arg > Stop TCL10A7 Breast G407A exon 5 136 Cys > Tyr TCL10F5Submaxillary Gland T455C exon 5 152 Leu > Pro TCL10H8 Leukemia C517Texon 6 173 Arg > Cys TCL10F7 Testis G518C exon 6 173 Arg > Pro TCL11F5Glioblastoma C697T exon 7 233 Arg > Stop BT-88 Breast^(1,2) 705 del Aexon 7 235 protein truncation TCL10A3 Breast 823 del G exon 7 275protein truncation¹Primary tumor specimens.²Analysis of corresponding normal DNA has shown that the TS10q23.3mutation of this primary breast tumor sample is somatic. Similaranalysis of the TS10q23.3 alterations in the other three primary tumorspecimens was not possible because corresponding normal DNAs were notavailable. The inventors have, however, determined that all nine primarytumor mutations previously observed by Steck et al., (1997) arosesomatically.

In addition to primary tumors, the inventors examined a set of tumorcell lines for alterations in the TS10q23.3 gene. These TCLs permittedthe inventors to investigate cancer types that were not represented inthe panel of primary tumors screened, including leukemia, lymphoma,neuroblastoma, retinoblastoma, as well as bladder, testis and uterinecancers. Out of the 75 TCLs exhibited LOH, the inventors identified tenhomozygous deletions that affected the coding regions of TS10q23.3 (FIG.13A and FIG. 13B). The homozygous deletions were present in TCLs fromastrocytomas (1/1), bladder carcinoma (1/3), breast carcinoma (1/14),glioblastoma (2/8), lung carcinoma (1/7), melanoma (4/7) and prostatecarcinoma (1/2). Whereas two of the cell lines had lost all nineTS10Q23.3 exons, the other eight TCLs had homozygously deleted differentcoding portions of the gene. Analysis of the remaining 65 TCLs revealedone frameshift, one nonsense and seven non-conservative missensevariants (Table 7).

Due to the relatively low frequency of observed TS10q23.3 mutations inprimary tumors compared to that observed in TCLs, the inventors examinedthe expression of TS10q23.3 in a series of ten GBM and ten normalspecimens. All normal samples exhibited expression of TS10q23.3, whilenone of the GBMs exhibited significant expression of this message (FIG.13C and FIG. 13D). Weak signals were observed in certain samples uponprolonged exposure, although the inventors could not distinguish whetherthese levels of message were detected due to contamination of normalcells in the sections or low TS10q23.3 expression within the tumorcells. However, this observation suggests that the altered expression ofTS10q23.3 may potentially play a role in the tumorigenesis of theseGBMs. The mechanism(s) of inhibition of TS10q23.3 expression and thelevel of TS10q23.3 expression in other types of primary tumors arecurrently under investigation.

The inventors have investigated a large panel of primary tumors andTCLs, prescreened for LOH, for alterations in TS10q23.3. In this set of84 primary tumors, the inventors only detected four potentialinactivating TS10q23.3 mutations. Taken together with the inventors'previous findings (Steck et al., 1997), 8/31 (26%) primaryglioblastomas, 3/31 (10%) primary breast, 1/8 (13%) primary kidney and1/11 (9%) primary melanoma tumors showed TS10q23.3 alterations.Interestingly, two of the five pediatric GBMs exhibited TS10q23.3alterations that should lead to the expression of non-functionalprotein, suggesting that further analysis of TS10q23.3 involvement inthis childhood disease is warranted. In the set of 75 TCLs, theinventors observed a total of 19 putative inactivating TS10q23.3mutations.

The actual incidence of TS10q23.3 mutations in the different cancertypes will likely be between the frequency observed in primary tumorsand that observed for the TCLs. The inventors' findings show that incomparison to primary tumors, TCLs harbor a significantly higherincidence of mutations in TS10q23.3 (Table 6). A similar observation hasbeen reported by Spruck et al. for mutations of p16 in bladder cancers(Spruck III, et al., 1994). This discrepancy is likely due to one ormore of the following possibilities. First, in order to be successfullycultured in vitro, tumor cells may require certain combinations ofgenetic lesions that are acquired in vivo. Second, mutation events inTS10q23.3 may confer a growth advantage or cause clonal selection duringthe passaging of TCLs in vitro. Third, the substantially reducedexpression of TS10q23.3 observed in 10/10 primary GBM specimens suggestthat certain tumors may not have coding mutations in this gene but mayinstead express diminished levels of functional TS10q23.3. And fourth,normal cell contamination and specimen heterogeneity of primary tumorsmay prevent the detection of homozygous deletions, a mutationalmechanism observed for a significant number of TCLs at the TS10q23.3locus. In control experiments, it was determined that even the presenceof 5% contaminating normal tissue DNA within the tumor samples willprevent the identification of homozygous deletions using theseprocedures. Thus, the presence of homozygous deletions affectingTS10q23.3 in primary tumors could easily be underestimated by theinventors' analysis and will require alternative approaches to evaluatetheir occurrence. However, an additional complication is the presence ofan apparently unspliced TS10q23.3 pseudogene, located on chromosome 9q;the coding sequence of TS10q23.3 differs from this putative pseudogenein 16/1209 bases (see Methods).

A compilation of TS10q23.3 alterations shows that the spectrum ofvariants is diverse (FIG. 14). All of the non-conservative missensesubstitutions identified are found in the N-terminal portion ofTS10q23.3 within its putative phosphatase domain. In contrast, thelesions that result in the truncation of TS10q23.3 are distributedthroughout the gene. If all of the truncated forms of TS10q23.3 arenonfucntional, then the data indicate that the carboxy-terminal regionof TS10q23.3 is essential for the expression of active protein. This isconsistent with the notion that the potential phosphorylation sites andPDZ motif are important for TS10q23.3 function. Alternatively, thesequences of the C-terminal region of this protein may be required forproper folding. Of interest, the only germline mutations in TS10q23.3reported to date have been detected in individuals with Cowden'ssyndrome (Liaw et al., 1997); all other primary tumor TS10q23.3 variantscharacterized have arisen somatically (Table 7). The diversity of theTS10q23.3 alterations observed predict that many distinct lesions ofthis gene exist in the population. Overall, the data suggest thatTS10q23.3 is a tumor suppressor that plays a significant role in thegenesis of many types of cancers.

Example 8 Role of TS10q23.3 Mutations in Early Onset Breast Cancer:Causative in Association with Cowden's Syndrome and Excluded inBrca1-Negative Cases

Methods

Clinical Materials: Blood samples were obtained after informed consentfrom individuals with Cowden's syndrome. An aliquot was used for DNAextraction, while peripheral blood mononuclear cells were purified froma second sample and used to generate an EBV-transformed lymphoblastoidcell line. The diagnosis of CS was made using the International Cowden'sConsortium CD diagnostic criteria (Nelen et al., 1996). For individualswith early onset breast cancer, the sample consists of 63 women whodeveloped breast cancer before age 35 (average age at diagnosis is 27.7yrs), did not have a clinical diagnosis of CS, and who had previouslybeen shown not to carry clearly deleterious mutations in BRCA1 (5 womenin the sample carried missense polymorphisms of unknown significance).These women are a subset of a sample of 798 unrelated individuals from20 collaborating institutions, chosen from families which were generallyat an elevated risk of carrying BRCA1 mutations. Most families werechosen because of multiple cases of breast cancer, early age of breastcancer diagnosis, and incidence of ovarian cancer, as these conditionshave been previously shown to be associated with germline mutations ofBRCA1. Some of the families extended to second degree relatives. Allsamples from institutions in the United States were collected fromindividuals participating in research studies on the genetics of breastcancer. A1 samples from institutions outside of the United States werecollected according to the appropriate guidelines concerning researchinvolving human subjects imposed by the institution's equivalentauthorities. Only one representative from each family was included inthe sample, and no families known to be linked by genetic markers toBRCA1 were included. This is a heterogeneous sample which represents thediversity amongst patients who present at high-risk clinics as opposedto the more controlled sampling done for family or population studies.This has directed the inventors' analyses towards methods which do notrequire that sample frequencies of subgroups reflect frequencies in thegeneral population. Therefore the inventors can assess, for example, theprobability that a woman with breast cancer diagnosed at age 30 carriesa deleterious BRCA1 or TS10q23.3 (also referred to as MMAC1) mutation,but the inventors cannot estimate the frequency of such women in thegeneral population. All the samples used in the TS10Q23.3 study werestripped of identifiers.

DNA Extraction: After informed consent was obtained, patients' genomicDNA was extracted from whole blood or lymphoblastoid cell lines usingQ1Aamp blood Maxi Kit. Concentration was measured by OD₂₅₀ and puritywas checked by the ratio of OD₂₆₀/OD₂₈₀.

Genotyping: Primer pairs for the chromosome 10 locus were obtained fromResearch Genetics. The forward strand primer was end-labeled in thepresence of ³³P-γATP and polynucleotide kinase. PCR™ reactions wereperformed in a total reaction volume of 30 microliters. The reactionsconsisted of 10 mM of each primer, 200 mM of deoxynucleotides, 1.5 unitsof Taq DNA polymerase and 50 ng of genomic DNA. PCR™ was performed for35 cycles with 45 seconds denaturation at 94° C., 45 seconds annealingat 55° C. and 1 min elongation at 72° C. A final 10 min elongation wasused. PCR™ reactions were stopped by addition of 20 microliters of stopsolution (95% formamide, 1 mM EDTA, 0.25% bromophenol blue, 0.25% xylenecyanol). Then reactions were denatured for 5 min at 94° C. and theproducts were separated on a 8% denaturing polyacrylamide gel. Allelesizes were determined by comparing to the SequaMark (Research Genetics)which was included as a size standard on the gels.

Linkage Analysis: Two-point linkage analysis was performed using MLINK.Individuals below 20 years were considered as unknown. Disease genefrequency was set equal to 0.000001 and marker allele frequencies wereestimated using ILINK. Both MLINK and ILINK are from the LINKAGE packageVersion 5.2 (Lathtop et al., 1984). Reconstruction of the most probablehaplotypes in family D was obtained using GENEHUNTER (Kruglyak et al.,1996). Pedigrees were drawn using Cyrillic Version 2.02.

Results

Cowden's syndrome (CS) (Lloyd and Dermis, 1963), or multiple hamartomasyndrome (Weary et al., 1972), is an autosomal dominant disorderassociated with the development of hamartomas and benign tumors in avariety of tissues, including the skin, the thyroid, the breast, thecolon and the brain. It has been suggested that women with CS are atincreased risk for breast cancer (Brownstein et al., 1978) and, as inother susceptibility syndromes, they appear to develop breast cancer atan early age. CS is also associated with a specific skin lesion, thetrichilemmoma (tumor of the follicular infundibulum), and thus thisbreast cancer susceptibility syndrome can be recognized by the presenceof a cutaneous biomarker (Brownstein et al., 1977; 1978). The inventorshave studied in detail the clinical and pathological findings in thissyndrome and have demonstrated that the mean age of presentation withmalignant breast disease in CS is 46 years, with the age range ofpresentation with breast cancer in affected women from 33 to 74 years.Moreover, very few of the women with CS that the inventors studied had afamily history of breast cancer. Of interest, men with CS appear not tobe at increased risk for the development of breast cancer (Brownstein etal., 1978). The inventors have also shown that women with CS developexuberant benign breast disease and frequently report a history ofmultiple breast biopsies prior to the development of breast cancer. Thehistory of skin disease and benign breast disease can therefore allowidentification of affected individuals prior to the development ofbreast cancer in this high risk population.

It has been previously demonstrated that a locus for CS exists onchromosome 10 (Nelen et al., 1996). In that study, a total of 12families were examined resulting in the identification of the Cowdencritical interval between markers D10S215 and D10S564. Certain affectedindividuals in these families had CS and Lhermette-Duclos disease (LDD)(Nelen et al., 1996; Liaw et al., 1997), a rare brain disordercharacterized by a dysplastic gangliocytoma of the cerebellum (Albrechtet al., 1992). Fine mapping of this area refined this initial result(Liaw et al., 1997), supporting a location for the CS gene betweenmarkers D10S215 and D10S541. More recently, affected individuals in fourfamilies with CS have been shown to have germline mutations (Liaw etal., 1997) in a gene known as PTEN (Li et al., 1997), TS10Q23.3 (Stecket al., 1997) or TEP1 (Li et al., 1997) which is located in the Cowdencritical interval on chromosome 10. Of interest, the predicted TS10q23.3protein contains sequence motifs with significant homology to thecatalytic domain of protein phosphatases, and to the cytoskeletalproteins, tensin and auxillin (Li et al., 1997; Steck et al., 1997).Moreover, coding region mutations in TS10q23.3 were observed in humantumors or tumor cell lines of the breast, brain, prostate and kidney (Liet al., 1997; Steck et al., 1997). While the function of this gene isunknown, it is likely that TS10q23.3 plays a role in the control of cellproliferation and its loss of function is important in the developmentof human tumors.

Linkage Analysis and Mutation Screening in CS Kindreds

In order to extend the observations indicating a CS locus on chromosome10, the inventors performed a two point linkage analysis using fivemarkers located in the Cowden critical interval, on four families withclinical evidence of CS (Nelen et al., 1996). All families were examinedin detail and the diagnosis of this syndrome was made using theinternational Cowden's Consortium CD diagnostic criteria (Nelen et al.,1996). Two small families displayed positive LOD scores that could notexclude linkage to three loci on chromosome 10 (see family A and B,Table 8). Two other families with clinical findings identical to thosedescribed above, showed significant negative lod-scores for some of themarkers in this region (families C and D, Table 8). A heterogeneity testalso was performed which gave non-significant results. These findingswere confirmed by the haplotypes construction (FIG. 15). In particular,in family C, individual 2 transmits to both her affected children thehaplotype inherited from her unaffected father. Finally, in family D,individuals 2 and 20 have inherited a haplotype different from one oftheir affected relatives. TABLE 8 Twopoint Analysis of CD Families withCA Repeat Markers 0.0 0.01 0.05 0.1 0.2 0.3 0.4 FAMILY A D10S579 0.000.00 0.00 0.00 0.00 0.00 0.00 D10S215 0.30 0.30 0.28 0.26 0.20 0.15 0.08D10S541 0.00 0.00 0.00 0.00 0.00 0.00 0.00 D10S1739 0.30 0.30 0.28 0.260.20 0.15 0.08 D10S564 0.30 0.30 0.28 0.26 0.20 0.15 0.08 FAMILY BD10S579 0.00 0.00 0.00 0.00 0.00 0.00 0.00 D10S215 0.30 0.29 0.26 0.210.13 0.06 0.02 D10S541 0.00 0.00 0.00 0.00 0.00 0.00 0.00 D10S1739 0.300.29 0.26 0.21 0.13 0.06 0.02 D10S564 0.30 0.29 0.26 0.21 0.13 0.06 0.02FAMILY C D10S579 0.00 0.00 0.00 0.00 0.00 0.00 0.00 D10S215 −infinity−3.40 −2.00 −1.40 −0.80 −0.44 −0.19 D10S541 0.00 0.00 0.00 0.00 0.000.00 0.00 D10S1739 −0.05 −0.06 −0.09 −0.13 −0.16 −0.15 −0.09 D10S564−infinity −3.40 −2.00 −1.40 −0.80 −0.44 −0.19 FAMILY D D10S579 −infinity−1.52 −0.28 0.11 0.28 0.19 0.05 D10S215 −infinity −1.58 −0.33 0.07 0.250.18 0.05 D10S541 −infinity −1.44 −0.39 0.01 0.22 0.18 0.06 D10S1739−2.20 −0.45 0.14 0.32 0.35 0.23 0.08 D10S564 −0.03 0.08 0.30 0.38 0.350.22 0.07

TABLE 9 Mutation Exon/Intron Predicted Effect 1. 79linsAT Exon 7Frameshift 2. 915del13 Exon 8 Frameshift 3. 137ins3 Exon 2 One aminoacid insertion (Asn)

Using a PCR™ and sequencing based approach, the inventors examined the 9exons and associated splice junctions of TS10Q23.3, using the describedprimers (Steck et al., 1997), in 16 affected individuals from these 4families. Of interest, 4 of these 16 individuals had breast cancer, and2 of the 4 had breast cancer prior to the age of 40. The inventorsfailed to detect mutations in the coding sequence in these 16individuals from these 4 families with the classic symptoms and signs ofCS.

Mutational Analysis in Individuals with CS

The inventors then screened a set of 31 affected individuals from 23families with CS whose kindreds had not been used in the inventors'linkage studies. Of the 31 individuals, 13 were related individuals from5 families. Thus, a total of 23 unrelated probands were screened. Asingle affected female (Walton et al., 1986) demonstrated a frameshiftmutation in exon 7 of the coding sequence (see FIG. 16). Specifically,the inventors demonstrated an AT insertion after nucleotide 791(791insAT), thus resulting in a frameshift and downstream prematuretermination codon. Of interest, this woman developed mammogram negativebreast cancer at the age of 36, which was discovered at the time ofprophylactic mastectomy (Walton et al., 1986). The proband had anunaffected brother, as well as an affected daughter. Direct sequencingof exon 7 in these individuals demonstrated the presence of theidentical mutation in the affected daughter (FIG. 16) and the absence ofthe mutation in the unaffected brother. In studying a second individualwith CS and early onset breast cancer (age 33), the inventorsdemonstrated a three base insertion in exon 2 (137ins3), resulting inthe insertion of a single amino acid (Asn). Finally, in another womanwith bilateral breast cancer and endometrial cancer, the inventorsidentified a 13 base pair frame shift deletion in exon 8 (915de112).These data demonstrate 3 more mutant alleles of TS10Q23.3 that areassociated with CS (Liaw et al., 1997), and in particular, with CS andbreast cancer (Brownstein et al., 1978). However, in 27 individuals from20 families, the inventors did not detect mutations in the codingsequences of TS10Q23.3. In this population, 7 of these individuals hadbreast cancer, although all of these women developed breast cancer afterthe age of 40. One of these 7 individuals had bilateral breast cancer.In total, therefore, combining the family data, as well as theseindividuals, the inventors detected coding sequence mutations in 4individuals from 3 CS families, but did not detect coding sequencealterations (i.e., missense or silent variants) in 43 other individualsfrom 24 families with CS.

Mutational Analysis in Women with Early Onset Breast Cancer

A strong case has been made for the existence of a genetic mechanismregulating breast tumor formation in early onset breast cancer (thedevelopment of breast cancer before the age of 40) (Claus et al., 1990).As CS is inherited in an autosomal dominant fashion, the geneticmechanisms regulating the development of breast cancer in thispopulation may also play a role in the development of early onset breastcancer. Since the inventors detected germline TS10Q23.3 mutations in CSassociated with early onset breast cancer, and mutations in this geneoccur at relatively high frequency in breast tumors and breast tumorcell lines (Steck et al., 1997, Li et al., 1997), the inventors wantedto further investigate the role of germline TS10Q23.3 mutations in earlyonset breast cancer. In an effort to bias the inventors towards a sampleset potentially enriched in germline TS10Q23.3 mutations, the inventorssequenced the gene in 63 women who developed breast cancer before age 35(average age at diagnosis 27.7 years), did not appear to have a clinicaldiagnosis of CS, and who had previously been shown not to carry dearlydeleterious mutations in BRCA1 (5 women in the sample carried missensepolymorphisms of unknown significance). No coding sequence alterationswere detected in the 9 exons of TS10Q23.3 in this sample set. Incontrast, using the exact same mutation detection and analysis criteriaon a similarly ascertained set of non-Ashkenazi breast cancer affecteds(without exclusion of BRCA1 carriers), the inventors would expect todetect 7 deleterious mutations and 5 missense polymorphisms of unknownsignificance in BRCA1. Furthermore, outside of the 4 CS patientscarrying germ line mutations in TS10Q23.3 described above, the inventorshave detected no sequence polymorphisms in the coding sequence of thisgene in more than 200 germline chromosomes, and in fact find only onesequence difference (silent) between the human and chimpanzee sequences.If the frequency of coding and proximal splice junction sequencevariants in TS10Q23.3 were 5% in the population from which this samplewas drawn, then the inventors would have had a 95% chance of detectingone or more such variant.

Discussion

Cowden's syndrome is distinct among autosomal dominant genetic syndromesthat predispose to the development of breast cancer as it has a uniquecutaneous biomarker, the trichilemmoma (Brownstein et al., 1997; 1978).Furthermore, women with CS frequently give a history of multiple breastbiopsies for benign breast disease prior to the development of breastcancer. Most of these women did not have a family history of breastcancer. To date, the most well described association of CS with organspecific cancer susceptibility is the female breast (Brownstein et al.,1977). Other organ systems that appear to develop cancer with increasedfrequency in these individuals such as the thyroid. In contrast to otherautosomal breast cancer susceptibility syndromes, such as the oneassociated with mutations in BRCA1 (Ford et al., 1995), the developmentof ovarian cancer in this syndrome is quite rare. However, CS shareswith these syndromes an earlier age of onset of breast cancer, as wellas an increased likelihood of bilateral breast cancer. Previousobservations demonstrated linkage of CS to chromosome 10q22-23 (Nelen etal., 1996). Furthermore, it is also now evident that mutations in a gene(Liaw et al., 1997) known as PTEN (Li et al., 1997), TS10Q23.3 (Steck etal., 1997) or TEP1 (Li and Sun, 1997) found in the Cowden's criticalinterval on chromosome 10, are associated with CS individuals (Liaw etal., 1997).

In the observations reported here, the inventors identify 3 new germlinemutations in the coding sequence of TS10Q23.3 associated with CS, andspecifically in individuals with CS and breast cancer. In two, relatedindividuals with CS, the inventors described a frameshift mutation inexon 7, resulting in a premature termination codon, that is identical inan affected mother and her affected daughter. This TS10Q23.3 mutationappears to be associated with early onset breast cancer, as one of thetwo affected individuals developed breast cancer at age 36. In a thirdaffected individual, the inventors identified a 13 base pair deletion inexon 8. While this individual did not develop breast cancer at an earlyage, she had a history of bilateral breast cancer. Of interest, she alsodeveloped endometrial cancer while on tamoxifen. Given that endometrialcancer has been associated with CS (Starink et al., 1986) and withtamoxifen use (Fornander et al., 1989), the contribution of both riskfactors to the development of disease in this one women is unknown.However, this raises the possibility that the subpopulation of women whodevelop endometrial cancer while on tamoxifen may have CS and/ormutations in TS10Q23.3. Finally, the inventors identified a 3 baseinsertion in exon 2 in a another woman who developed breast cancer atthe age of 33.

In the set of CS individuals that the inventors studied, the inventorsdetected germline TS10Q23.3 mutations in 4 individuals from 3 families,but did not observe any coding sequence alterations in the remaining 43individuals from 24 unrelated families. These data supported theinventors' limited linkage information, suggesting that all CS familiesmay not link to the locus identified on chromosome 10. While the studiesthe inventors performed do not rule out mutations in the 5′ regulatoryregions or in the 3′ untranslated region of TS10Q23.3, or othermechanisms that alter its expression level, such as methylationsilencing, as being associated with CS, both the linkage data and theDNA sequencing results support the idea that the CS may be geneticallyheterogeneous. Tuberous sclerosis, another autosomal dominant disorderassociated with the formation of hamartomas in the skin and otherorgans, has been shown to be genetically heterogeneous with distinctloci located at chromosome 9q34 (Haines et al., 1991) and chromosome16p13.3 (Kandt et al., 1992). The inventors' results indicate that thisalso may be true for CS. Why this was not demonstrated in the initialobservations is not clear, but could be due to the ethnic backgrounds ofthe initial families examined (Nelen et al., 1996; Liaw et al., 1997).Moreover, certain of these individuals presented with CS andLhermette-Duclos disease, which the inventors have never seen in a CSproband or in a CS family (Nelen et al., 1996; Liaw et al., 1997).

A strong case has been made for the existence of a genetic mechanismregulating breast tumor formation in early onset breast cancer (Claus etal., 1990). Indeed, early onset breast cancer has been associated withmutations in the BRCA1 (Miki et al., 1994) and BRCA2 (Wooster et al.,1995). CS is associated with early onset breast cancer, and the canceris usually ductal carcinoma (Brownstein et al., 1977; Brownstein et al.,1978). Rachel Cowden, for whom the syndrome is named, apparently died ofbreast cancer at age 31 (Lloyd and Dennis, 1963; Brownstein et al.,1978). As described herein, the inventors have identified TS10Q23.3mutations in 2 CS individuals with early onset breast cancer, as well asin 1 with bilateral breast cancer. However, when the inventors searchedfor germline TS10Q23.3 mutations in a subgroup of women with early onsetbreast cancer, lacking the signs of CS and previously shown to havewild-type sequences of BRCA1, the inventors failed to detect anysequence variants. These data suggest that germline mutations inTS10Q23.3 occur infrequently in at least this subpopulation of earlyonset breast cancer cases.

Example 9 Suppression of Tumorigenicity of Glioblastoma Cells byAdenovirus-Mediated MMAC1/PTEN Gene Transfer

Additional studies were designed to further evaluate the function ofMMAC1/PTEN as a tumor suppressor. A replication-defective adenovirus(MMCB) was constructed for efficient, transient transduction of MMAC1into tumor cells. The data presented in this Example support an in vivotumor suppression activity of MMAC1/PTEN, and suggests that in vivo genetransfer with this recombinant adenoviral vector will be useful incancer gene therapy.

Materials and Methods

Cell Lines: The MMAC1-mutated glioblastoma cell line U87MG was obtainedfrom the American Type Culture Collection (ATCC). Cells were maintainedin culture medium (DME/10% FBS/1% L-glutamine) in a humidifiedatmosphere containing 7% CO₂ at 37° C. 293 embryonic kidney cells werealso obtained from ATCC and were grown in DME culture mediumsupplemented with 10% FBS.

RT-PCR analysis: Total RNA was isolated from U87MG cells (Tri Reagent,Molecular Research Center) per manufacturer's instructions. RNA wasreverse-transcribed using MuLV-RT (RNA PCR kit, Perkin-Elmer), randomhexamer and other kit reagents, followed by PCR using primers MAC1.6f(5′-CTG CAG AAA GAC TTG AAG GCG TA-3′, SEQ ID NO:58) and MAC1.6r (5′-GCCCCG ATG TAA TAA ATA TGC AC-3′) (SEQ ID NO:59) matching sequences inMMAC1 exons 2 and 5, respectively. Amplification conditions were 95° C.denaturation for 1 min, then (95° C., 15″; 55° C., 30″) for 25 cycles,then 72° C. for 5 min. The expected normal product size was 317 bp. Theabnormal band from U87MG was cut out from an agarose gel, purified(UltraClean, Mo Bio Labs), and directly sequenced using an automatedsequencing system (ABI 373A, Perkin Elmer).

Viruses: A recombinant adenovirus containing wild-type p53 (FTCB) wasconstructed as described previously (Wills et al., 1994). The genome ofthis vector has deletions of the E1 and E3 regions and protein 1× gene,and expresses its transgene under control of the human cytomegalovirus(CMV) immediate early promoter/enhancer. The MMAC1/PTEN vector MMCB wasconstructed in exactly the same manner except that p53 was replaced witha cDNA encoding full-length MMAC1 (Steck et al., 1997). The controlvector GFCB was constructed to match MMCB except for its transgene,enhanced green fluorescent protein (Clontech). Another matching controlvector, ZZCB, was constructed without a transgene. The BGCA controlvector expressing E. coli LacZ driven by the CMV promoter wasconstructed in a genome with partial E4 deletion in addition todeletions of E1, E3, and protein IX (Wang et al., 1997) because ofpackaging size constraints. All viruses were grown in 293 cells andpurified by DEAE column chromatography as described (Huyghe et al.,1995). Virus particle concentrations were determined by Resource Q HPLC(Shabram et al., 1997), and the primary structure of all transgenes wasverified by automated sequencing of viral DNA.

Immunodetection of MMAC1 protein: Cell monolayers were infected for 24hr with GFCB or MMCB at various viral particle numbers per milliliter ofgrowth medium (pn/ml). Virus-containing solutions were removed at 24 hrand cells were either harvested at this time or refed with growth mediumand collected at later time points. Cells were harvested by scrapinginto cold phosphate-buffered saline (PBS), centrifuged and washed oncemore in cold PBS, then freeze-thawed and resuspended in lysis buffer [50mM MOPS. pH 7.0, 150 mM NaCl, 1% NP-40, 5% glycerol, 0.4 mM EDTA andsupplemented with 1 mM DTT and 1× Complete Protease Inhibitor Cocktail(Boehringer Mannheim)]. Cell lysates were clarified by centrifugation at10,000×g for 15 min, and supernatants were normalized for proteincontent. Samples were resolved by SDS-PAGE using pre-cast 8%TRIS-glycine gels (Novex), then transferred to poly(vinylidenedifluoride) membranes (Immobilon-P) for Western blotting. Membranes wereblocked with TBST containing 5% skim milk, and then blotted withanti-MMAC1 rabbit polyclonal antibody (BL74), followed by donkey andrabbit IgG conjugated with horseradish peroxidase (Amersham). MMAC1 wasdetected by chemiluminescence (HCL kit, Pierce) using Kodak XAR-5 film.

FACS infectivity assay: U87MG cells were plated at 2×10⁵ cells/well in6-well plates and incubated overnight, then infected with GFCB atconcentrations ranging from 1×10⁵ to 1×10⁹ particles/ml for 24 hr. Cellswere harvested by trypsinization and assayed by flow cytometry (BectonDickenson FACScan) for green fluorescence (525 nm peak detection, filterFL-I). Cells were gated on forward and side scatter, and a cutoff offluorescence intensity was established such that ˜99% of uninfectedcells were negative. The percentage of GFCB-infected cells with greaterfluorescence than this cutoff was then determined, representing aminimum estimate of the percentage of infected cells.

³H-thymidine incorporation assay: Cells were plated at 5×10³ cells/wellin 96-well microtiter plates (Costar) and incubated overnight. Dilutionsof ZZCB, GFCB, FTCB and MMCB in medium ranging from 5×10⁶ to 1×10⁹particles/ml were added in triplicate to the cell monolayers and thenincubated for 24 hr. Virus-containing solutions were removed at 24 hrafter infection and replaced with new tissue culture medium for anadditional 24 hr. Cells were treated with 1 μCi of ³H-thymidine per well4 hr prior to harvesting. Cell were harvested onto glass-fiber filters,and incorporation of ³H-thymidine was determined using liquidscintillation (Top Count, Packard). Results are plotted as percentagesof buffer-treated control (mean±SD).

Cell count/viability assay: Subconfluent monolayers of U87MG cells wereinfected in triplicate with MMCB or GFCB adenovirus at variousconcentrations for 24 hr, after which supernatants was replaced withfresh tissue culture medium for 48 addition hr. Cells were thenharvested by trypsinization, and viable cells were counted by the trypanblue exclusion method using a hemocytometer.

Soft agar colony formation assay: U87MG cells infected as above with5×10⁶, 5×10⁷ or 5×10⁸ particles/fill for 24 hr were suspended in tissueculture medium containing 0.35% agar and layered in triplicate onto 0.7%agar in 35 mm tissue culture wells. Cultures were incubated in ahumidified atmosphere containing 7% CO₂ at 37° C. with overlying tissueculture medium that was replaced every five days. Colony growth wasassessed at 14 days post infection.

Tumorigenicity assay: U87MG cells were plated at a density of 1×10⁷cells per T225 flask. After overnight incubation, cell monolayers wereinfected with 5×10⁷ or 5×10⁸ particles/ml of adenoviruses GFCB, FTCB,BGCA or MMCB for 24 hr. Infected or uninfected cells were harvested bytrypsinization, washed in medium, counted in the presence of TrypanBlue, and injected subcutaneously (5×10⁶ viable cells per flank) intoathymic nu/nu female mice (Simonsen Labs). Mice were scored for tumorsat 21 or 30 days; tumor diameters in 3 dimensions were measured withVernier calipers, and tumor volumes were calculated as their product.

Results and Discussion

U87MG human glioblastoma cells (Ponten and Macintyre, 1968) were chosenfor study based on their reported MMAC1 mutation (Steck et al., 1997),soft agar colony-forming ability and subcutaneous tumorigenicity in nudemice. An abnormally small RT-PCR product derived from U87MG RNA usingprimers in exons 2 and 5 (see Methods section above in Example 9) wasfound to lack exon 3 by sequencing, in agreement with the intron 3splice donor site mutation (Steck et al., 1997). Although exon 3contains 45 bp (15 codons) and an in-frame readthrough product ispossible, the missing residues encode a conserved alpha helix in thenative protein, and their loss ablated growth-inhibitory activity asmeasured by Fumari et al., (1997).

The purified recombinant MMAC1-containing adenovirus (MMCB) wascharacterized for transgene expression in U87MG cells by Westernblotting of cell lysates with a rabbit polyclonal antibody (FIG. 17).Endogenous MMAC1 protein was not detected in uninfected or controlvirus-infected cells, but was detected in a dose-dependent fashion inMMCB-infected cells by the end of the 24 hr infection period, as well asat 48 hr, 72 hr and 96 hr (FIG. 17). This study verified the efficienttransduction and acute expression of exogenous MMAC1 protein in U87MGglioma cells as well as validating its detection by Western blottingwith antibody BL74.

Infectivity of U87MG cells was assessed quantitatively by FACS analysisusing a recombinant adenovirus identical to MMCB except for itstransgene, which encoded green fluorescent protein (FIG. 18). Theexpected sigmoidal infectivity curve was obtained, from which it wasestimated that 85-90% of cells were infected at a viral dose of 5×10⁷particles/ml for 24 hr. Of note is that the dosing parameters usedherein are not based on the plaque-forming unit or its derivative,multiplicity of infection, it has previously been shown that adenoviralconcentration and infection time are the primary determinants of invitro transduction (Nyberg-Hoffman et al., 1997).

In vitro proliferation of MMCB vs. control-Ad infected U87MG cells wasmeasured by ³H-thymidine uptake over a range of viral concentrations(FIG. 19A), U87MG was differentially inhibited by MMCB compared to twocontrol adenoviruses (GFCB and ZZCB) over most viral doses; at highadenovirus concentrations (e.g. 1×10⁹ particles/ml), a nonspecificinhibitory effect predominated, as has been noted before in some celllines (Harris et al., 1995). Inhibition of DNA synthesis by MMCB wascomparable to that induced by adenoviral p53 gene transfer (FTCB; FIG.19A).

Growth inhibition was confirmed in a second in vitro assay by countingviable cells at 72 hr after the start of infection (FIG. 19B), MMCBreduced cell numbers at this time point by about 50% compared to GFCB atequal doses. This inhibition was comparable in magnitude to thatobserved using transient plasmid transfection (Furnari et al., 1997).MMCB and GFCB infected cultures had similar viability rates at 72 hr andmorphological evidence of cell death, such as cell blebbing or nuclearfragmentation, was not seen with MMCB treatment.

Effects of MMAC1 on anchorage-independent growth were assessed as wellby colony formation in soft agar following transduction by MMCB vs. GFCBor FTCB. The latter was included in order to validate the assay with anestablished tumor suppressor gene. At a dose of 5×10⁷ particles/ml for24 hr, colony formation with MMCB or FTCB was inhibited by approximately50% compared to the GFCB control, whereas a >85% inhibition (relative toGFCB) could be achieved at 5×10⁸/ml of either MMCB or FFCB (FIG. 20).Therefore, a dose-dependent, gene-specific effect of MMAC1 was evidentin this in vitro assay.

Two tumorigenicity assays were performed with 5×10⁶ MMCB-infected U87MGcells per injection compared to the same number of cells infected bythree different control Ads: GFCB (green fluorescent protein in matchingΔEI/ΔE3 background), FTCB (p53 in matching ΔEI/ΔE3 background), and BGCA(LacZ in ΔE1/ΔE3/ΔE4 background) (Table 10). Differences betweenexperiments 1 and 2 included the use of two dose levels vs. one, andtermination at 21 vs. 30 days, respectively. MMCB-infected U87 cellswere completely nontumorigenic at 21 or 30 days with the exception ofthree very small tumors (˜10 mm³) at the lower dose level inExperiment 1. Tumors formed in all 39 mice injected with uninfected orcontrol-Ad infected cells. Reporter gene-containing control-Ads, GFCBand BGCA, had some activity in reducing average tumor size compared tobuffer-treated cells, a nonspecific “adenoviral effect” previously notedby the inventors (Wills et al., 1994; Harris et al., 1995). Thep53-containing Ad had a more dramatic effect on average tumor size (˜68mm³), yet tumors still formed in 6 of 6 mice. These results areconsistent with the growth-inhibitory effects of p53 adenovirus genetransfer in U87MG cells reported elsewhere, even though these cellscontain p53 alleles with the wild-type sequence (Gomez-Manzani et al.,1996; Kock et al., 1996). In any case, these data indicate agene-specific tumor suppression activity of MMCB in U87MG cells atmoderate viral doses.

Using a recombinant adenoviral gene transfer system, an in vitro growthinhibition activity of MMAC1/PTEN in U87MG cells was shown. The use of arecombinant adenovirus was helpful in circumventing the known technicaldifficulty of studying tumor cells stably expressing potentiallygrowth-inhibiting proteins such as MMAC1. A specific tumor suppressionactivity of MMAC1 was most clearly detected in the in vivo assay,supporting the importance of the tumorigenicity assay in determiningtumor suppression function. These data support a role for MMAC1inactivation in glioblastoma tumorigenesis, and further suggest thatMMAC1/PTEN gene transfer in vivo may be considered as a potential cancertherapy approach.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the compositions and/ormethods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1. A method for determining the presence or absence of PTEN in a cancercell comprising the steps of (i) obtaining a sample from a subject; and(ii) determining the expression of a functional PTEN in said cancer cellof said sample.
 2. The method of claim 1 wherein said cancer is selectedfrom brain, lung, liver, spleen, kidney, lymph node, small intestine,pancreas, blood cells, colon, stomach, breast, endometrial, prostate,testicle, ovary, skin, head and neck, esophagus, bone marrow and bloodcancer.
 3. The method of claim 1 wherein said cancer is prostate cancer.4. The method of claim 1 wherein said cancer is brain cancer.
 5. Themethod of claim 1 wherein said cancer is lung cancer.
 6. The method ofclaim 1 wherein said cancer is endometrial cancer.
 7. The method ofclaim 1 wherein said cancer is colon cancer.
 8. The method of claim 1wherein said cancer is head and neck cancer.
 9. The method of claim 1wherein said cancer is breast cancer
 10. The method of claim 1 whereinsaid sample is a tissue or fluid sample.
 11. The method of claim 1wherein said determining comprises assaying for a PTEN nucleic acid insaid sample.
 12. The method of claim 11 further comprising subjectingthe sample to conditions suitable to amplify said PTEN nucleic acid. 13.The method of claim 1 wherein said determining step comprises evaluatingthe structure of the PTEN gene, protein, or transcript.
 14. The methodof claim 13 wherein said evaluating the structure of the PTEN gene,protein, or transcript comprises sequencing, wild-type oligonucleotidehybridization, mutant oligonucleotide hybridization, SSCP™, or RNaseprotection.
 15. The method of claim 13 wherein said evaluating thestructure of PTEN gene, protein, or transcript comprises wild-type ormutant oligonucleotide hybridization, wherein the oligonucleotide isconfigured in an array on a chip or wafer.
 16. The method of claim 1wherein said determining step comprises determining the DNA sequence ofPTEN in said sample.
 17. The method of claim 1 wherein said determiningstep comprises contacting said sample with an antibody that bindsimmunologically to PTEN.
 18. The method of claim 17 wherein saidantibody is a monoclonal antibody.
 19. The method of claim 1 whereinsaid determining step comprises immunohistochemical detection of PTEN intissue sections.
 20. The method of claim 19 wherein said tissue sectionis from a tumor sample.
 21. The method of claim 20 wherein said tumorsample is selected from brain, lung, liver, spleen, kidney, lymph node,small intestine, pancreas, blood cells, colon, stomach, breast,endometrial, prostate, testicle, ovary, skin, head and neck, esophagus,bone marrow, and blood cancer.
 22. The method of claim 20 wherein saidtumor sample is selected from brain, lung, colon, stomach, breast,endometrial, prostate, and head and neck tumor sample.
 23. The method ofclaim 16 wherein said tumor sample is a brain cancer tumor sample. 24.The method of claim 16 wherein said tumor sample is a lung cancer tumorsample.
 25. The method of claim 16 wherein said tumor sample is a coloncancer tumor sample.
 26. The method of claim 16 wherein said tumorsample is a breast cancer tumor sample.
 27. The method of claim 16wherein said tumor sample is a prostate cancer tumor sample.
 28. Themethod of claim 16 wherein said tumor sample is an endometrial cancertumor sample.
 29. The method of claim 16 wherein said tumor sample is ahead and neck cancer tumor sample.
 30. The method of claim 1 whereinsaid determining step comprises comparing the expression of PTEN in acancer sample with the expression of PTEN in a non-cancer sample.
 31. Amethod for determining the presence or absence of PTEN in a cancer cellcomprising the steps of (i) obtaining a sample from a subject; (ii)contacting said sample with an antibody that binds immunologically toPTEN; and (iii) detecting the binding of said antibody to said PTEN. 32.The method of claim 31 wherein said cancer is selected from brain, lung,liver, spleen, kidney, lymph node, small intestine, pancreas, bloodcells, colon, stomach, breast, endometrial, prostate, testicle, ovary,skin, head and neck, esophagus, bone marrow, and blood cancer.
 33. Themethod of claim 31 wherein said cancer is prostate cancer.
 34. Themethod of claim 31 wherein said cancer is brain cancer.
 35. The methodof claim 31 wherein said cancer is lung cancer.
 36. The method of claim31 wherein said cancer is endometrial cancer.
 37. The method of claim 31wherein said cancer is colon cancer.
 38. The method of claim 31 whereinsaid cancer is head and neck cancer.
 39. The method of claim 31 whereinsaid cancer is breast cancer.
 40. The method of claim 31 wherein saidsample is a tissue or fluid sample.
 41. The method of claim 31 whereinsaid antibody is a monoclonal antibody.
 42. The method of claim 41wherein said monoclonal antibody comprises a detectable label.
 43. Themethod of claim 42 wherein said detectable label is selected from afluorescent label, a chemiluminescent label, a radiolabel, or an enzymelabel.
 44. A method for determining if both alleles of PTEN in a sampleare altered comprising (a) obtaining a sample from a patient; (b)determining if the sample has lost one allele of PTEN; and (c)determining if the sample has an alteration in the other allele of PTEN,wherein if said sample has lost one allele and the other allele isaltered then both alleles of PTEN are altered.
 45. The method of claim44 wherein said sample comprises a cancer cell.
 46. The method of claim45 wherein said cancer cell is brain, lung, liver, spleen, kidney, lymphnode, small intestine, pancreas, blood cells, colon, stomach, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,bone marrow, or blood cancer cell.
 47. The method of claim 44 whereinsaid sample is a tumor specimen.
 48. The method of claim 47 wherein saidtumor specimen is a brain, lung, liver, spleen, kidney, lymph node,small intestine, pancreas, blood cells, colon, stomach, breast,endometrium, prostate, testicle, ovary, skin, head and neck, esophagus,or bone marrow tumor specimen.
 49. The method of claim 44 wherein saidtumor specimen is a prostate cancer tumor specimen.
 50. The method ofclaim 44 wherein said tumor specimen is a brain cancer tumor specimen.51. The method of claim 44 wherein said tumor specimen is a lung cancertumor specimen.
 52. The method of claim 44 wherein said tumor specimenis an endometrial cancer tumor specimen.
 53. The method of claim 44wherein said tumor specimen is a colon cancer tumor specimen.
 54. Themethod of claim 44 wherein said tumor specimen is a head and neck cancertumor specimen.
 55. The method of claim 44 wherein said tumor specimenis a breast cancer tumor specimen
 56. A method for detecting a PTENalteration comprising the steps of (i) obtaining a sample from asubject; and (ii) determining if PTEN is altered in said sample.
 57. Themethod of claim 56 wherein said sample is a tumor sample.
 58. The methodof claim 57 wherein said tumor sample is a brain, lung, liver, spleen,kidney, lymph node, small intestine, pancreas, blood cells, colon,stomach, breast, endometrial, prostate, testicle, ovary, skin, head andneck, esophagus, or bone marrow tumor sample.
 59. The method of claim 57wherein said tumor sample is a prostate cancer tumor sample.
 60. Themethod of claim 57 wherein said tumor sample is a brain cancer tumorsample.
 61. The method of claim 57 wherein said tumor sample is a lungcancer tumor sample.
 62. The method of claim 57 wherein said tumorsample is an endometrial cancer tumor sample.
 63. The method of claim 57wherein said tumor sample is a colon cancer tumor sample.
 64. The methodof claim 57 wherein said tumor sample is a head and neck cancer tumorsample.
 65. The method of claim 57 wherein said tumor sample is a breastcancer tumor sample.
 66. The method of claim 57 wherein said PTENalteration comprises an alteration selected from a deletion, aninsertion, a frameshift, and a point mutation.